Transaminases and aminotranferases

ABSTRACT

Thermostable transaminase and aminotransferase enzymes derived from various ammonifex, aquifex and pyrobaculum organisms are disclosed. The enzymes are produced from native or recombinant host cells and can be utilized in the pharmaceutical, agricultural and other industries.

[0001] This application is a Continuation of U.S. patent application Ser. No. 08/599,171 filed on Feb. 9, 1996.

[0002] This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production and isolation of such polynucleotides and polypeptides. More particularly, the polynucleotides and polypeptides of the present invention have been putatively identified as transaminases and/or aminotransferases. Aminotransferases are enzymes that catalyze the transfer of amino groups from α-amino to α-keto acids. They are also called transaminases.

[0003] The α-amino groups of the 20 L-amino acids commonly found in proteins are removed during the oxidative degradation of the amino acids. The removal of the α-amino groups, the first step in the catabolism of most of the L-amino acids, is promoted by aminotransferases (or transaminases). In these transamination reactions, the α-amino group is transferred to the α-carbon atom of α-ketoglutarate, leaving behind the corresponding α-keto acid analog of the amino acid. There is no net deamination (i.e., loss of amino groups) in such reactions because the α-ketoglutarate becomes aminated as the α-amino acid is deaminated. The effect of transamination reactions is to collect the amino groups from many different amino acids in the form of only one, namely, L-glutamate. The glutamate channels amino groups either into biosynthetic pathways or into a final sequence of reactions by which nitrogenous waste products are formed and then excreted.

[0004] Cells contain several different aminotransferases, many specific for α-ketoglutarate as the amino group acceptor. The aminotransferases differ in their specificity for the other substrate, the L-amino acid that donates the amino group, and are named for the amino group donor. The reactions catalyzed by the aminotransferases are freely reversible, having an equilibrium constant of about 1.0 (ΔG^(0′)≈0 kJ/mol).

[0005] Aminotransferases are classic examples of enzymes catalyzing bimolecular ping-pong reactions. In such reactions the first substrate must leave the active site before the second substrate can bind. Thus the incoming amino acid binds to the active site, donates its amino group to pyridoxal phosphate, and departs in the form of an α-keto acid. Then the incoming α-keto acid is bound, accepts the amino group from pyridoxamine phosphate, and departs in the form of an amino acid.

[0006] The measurement of alanine aminotransferase and aspartate aminotransferase levels in blood serum is an important diagnostic procedure in medicine, used as an indicator of heart damage and to monitor recovery from the damage.

[0007] The polynucleotides and polypeptides of the present invention have been identified as transaminases and/or aminotransferases as a result of their enzymatic activity.

[0008] In accordance with one aspect of the present invention, there are provided novel enzymes, as well as active fragments, analogs and derivatives thereof.

[0009] In accordance with another aspect of the present invention, there are provided isolated nucleic acid molecules encoding the enzymes of the present invention including mRNAs, cDNAs, genomic DNAs as well as active analogs and fragments of such enzymes.

[0010] In accordance with another aspect of the present invention there are provided isolated nucleic acid molecules encoding mature polypeptides expressed by the DNA contained in ATCC Deposit No. ______.

[0011] In accordance with yet a further aspect of the present invention, there is provided a process for producing such polypeptides by recombinant techniques comprising culturing recombinant prokaryotic and/or eukaryotic host cells, containing a nucleic acid sequence of the present invention, under conditions promoting expression of said enzymes and subsequent recovery of said enzymes.

[0012] In accordance with yet a further aspect of the present invention, there is provided a process for utilizing such enzymes, or polynucleotides encoding such enzymes for transferring an amino group from an α-amino acid to an α-keto acid. Most transaminases use L-amino acids as substrates, but as described below, it is also possible to convert the transaminases of the invention to use D-amino acids as substrates, thereby increasing their array of uses to include, for example, manufacture of synthetic pyrethroids and as components of β-lactam antibiotics. The transaminases of the invention are stable at high temperatures and in organic solvents and, thus, are superior for use with L- and/or D-amino acids for production of optically pure chiral compounds used in pharmaceutical, agricultural and other chemical industries.

[0013] In accordance with yet a further aspect of the present invention, there are also provided nucleic acid probes comprising nucleic acid molecules of sufficient length to hybridize to a nucleic acid sequence of the present invention.

[0014] In accordance with yet a further aspect of the present invention, there is provided a process for utilizing such enzymes, or polynucleotides encoding such enzymes, for in vitro purposes related to scientific research, for example, to generate probes for identifying similar sequences which might encode similar enzymes from other organisms by using certain regions, i.e., conserved sequence regions, of the nucleotide sequence.

[0015] These and other aspects of the present invention should be apparent to those skilled in the art from the teachings herein.

[0016] The following drawings are illustrative of embodiments of the invention and are not meant to limit the scope of the invention as encompassed by the claims.

[0017]FIG. 1 is an illustration of the full-length DNA (SEQ ID NO:17) and corresponding deduced amino acid sequence (SEQ ID NO:25) of Aquifex aspartate transaminase A of the present invention. Sequencing was performed using a 378 automated DNA sequencer (Applied Biosystems, Inc.) for all sequences of the present invention.

[0018]FIG. 2 is an illustration of the full-length DNA (SEQ ID NO:18) and corresponding deduced amino acid sequence (SEQ ID NO:26) of Aquifex aspartate aminotransferase B.

[0019]FIG. 3 is an illustration of the full-length DNA (SEQ ID NO:19) and corresponding deduced amino acid sequence (SEQ ID NO:27) of Aquifex adenosyl-8-amino-7-oxononanoate aminotransferase.

[0020]FIG. 4 is an illustration of the full-length DNA (SEQ ID NO:20) and corresponding deduced amino acid sequence (SEQ ID NO:28) of Aquifex acetylornithine aminotransferase.

[0021]FIG. 5 is an illustration of the full-length DNA (SEQ ID NO:21) and corresponding deduced amino acid sequence (SEQ ID NO:29) of Ammonifex degensii aspartate aminotransferase.

[0022]FIG. 6 is an illustration of the full-length DNA (SEQ ID NO:22) and corresponding deduced amino acid sequence (SEQ ID NO:30) of Aquifex glucosamine:fructose-6-phosphate aminotransferase.

[0023]FIG. 7 is an illustration of the full-length DNA (SEQ ID NO:23) and corresponding deduced amino acid sequence (SEQ ID NO:31) of Aquifex histidinol-phosphate aminotransferase.

[0024]FIG. 8 is an illustration of the full-length DNA (SEQ ID NO:24) and corresponding deduced amino acid sequence (SEQ ID NO:32) of Pyrobacullum aerophilum branched chain aminotransferase.

[0025]FIG. 9 is a diagramatic illustration of the assay used to assess aminotransferase activity of the proteins using glutamate dehydrogenase.

[0026] The term “gene” means the segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons).

[0027] A coding sequence is “operably linked to” another coding sequence when RNA polymerase will transcribe the two coding sequences into a single mRNA, which is then translated into a single polypeptide having amino acids derived from both coding sequences. The coding sequences need not be contiguous to one another so long as the expressed sequences ultimately process to produce the desired protein.

[0028] “Recombinant” enzymes refer to enzymes produced by recombinant DNA techniques; i.e., produced from cells transformed by an exogenous DNA construct encoding the desired enzyme. “Synthetic” enzymes are those prepared by chemical synthesis.

[0029] A DNA “coding sequence of” or a “nucleotide sequence encoding” a particular enzyme, is a DNA sequence which is transcribed and translated into an enzyme when placed under the control of appropriate regulatory sequences.

[0030] In accordance with an aspect of the present invention, there are provided isolated nucleic acids (polynucleotides) which encode for the mature enzymes having the deduced amino acid sequences of FIGS. 1-8 (SEQ ID NOS:17-32).

[0031] In accordance with another aspect of the present invention, there are provided isolated polynucleotides encoding the enzymes of the present invention. The deposited material is a mixture of genomic clones comprising DNA encoding an enzyme of the present invention. Each genomic clone comprising the respective DNA has been inserted into a pQE vector (Quiagen, Inc., Chatsworth, Calif.). The deposit has been deposited with: the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852, USA, on Dec. 13, 1995 and assigned ATCC Deposit No. ______.

[0032] The deposit(s) have been made under the terms of the Budapest Treaty on the International Recognition of the deposit of micro-organisms for purposes of patent procedure. The strains will be irrevocably and without restriction or condition released to the public upon the issuance of a patent. These deposits are provided merely as convenience to those of skill in the art and are not an admission that a deposit would be required under 35 U.S.C. §112. The sequences of the polynucleotides contained in the deposited materials, as well as the amino acid sequences of the polypeptides encoded thereby, are controlling in the event of any conflict with any description of sequences herein. A license may be required to make, use or sell the deposited materials, and no such license is hereby granted.

[0033] The polynucleotides of this invention were originally recovered from genomic DNA libraries derived from the following organisms:

[0034] Aquifex VF5 is a Eubacteria which was isolated in Vulcano, Italy. It is a gram-negative, rod-shaped, strictly chemolithoautotrophic, marine organism which grows optimally at 85-90° C. (T_(max)=95° C.) at pH 6.8 in a high salt culture medium with Q as a substrate, and H₂/CO₂+0.5% O₂ in gas phase.

[0035]Ammonifex degensii KC4 is a new Eubacaterial organism isolated in Java, Indonesia. This Gram negative chemolithoautotroph has three respiration systems. The bacterium can utilize nitrate, sulfate, and sulfur. The organism grows optimally at 70° C., and pH 7.0, in a low salt culture medium with 0.2% nitrate as a substrate and H₂/CO₂ in gas phase.

[0036]Pyrobaculum aerophilium IM2 is a thermophilic sulfur archaea (Crenarchaeota) isolated in Ischia Maronti, Italy. It is a rod-shaped organism that grows optimally at 100° C. at pH 7.0 in a low salt culture medium with nitrate, yeast extract, peptone, and O₂ as substrates and N₂/CO₂, O₂ in gas phase.

[0037] Accordingly, the polynucleotides and enzymes encoded thereby are identified by the organism from which they were isolated, and are sometimes hereinafter referred to as “VF5/ATA” (FIG. 1 and SEQ ID NOS:17 and 25), “VF5/AAB” (FIG. 2 and SEQ ID NOS:18 and 26), “VF5/A87A” (FIG. 3 and SEQ ID NOS:19 and 27), “VF5/AOA” (FIG. 4 and SEQ ID NOS:20 and 28), “KC4/AA” (FIG. 5 and SEQ ID NOS:21 and 29), “VF5/GF6PA” (FIG. 6 and SEQ ID NOS:22 and 30), “VF5/HPA” (FIG. 7 and SEQ ID NOS:23 and 31) and “IM2/BCA” (FIG. 8 and SEQ ID NOS:24 and 32).

[0038] The polynucleotides and polypeptides of the present invention show identity at the nucleotide and protein level to known genes and proteins encoded thereby as shown in Table 1. TABLE 1 Protein Protein DNA Gene w/closet Similarity Identity Identity Enzyme Homology (Organism) (%) (%) (%) VF5/ATA Bacillus subtilis 57.5 38.3 50.1 VF5/AAB Sulfolobus solfataricus 62.5 33.0 50.1 VF5/A87A Bacillus sphaericus BioA 67.4 42.9 51   VF5/AOA Bacillus subtilis argD 70.6 48.7 52.0 KC4/AA Bacillus YM-2 aspC 72.6 52.7 52.0 VF5/GF6PA Rhizobium 66.3 47.7 51.0 Leguminosarum NodM VF5/HPA Bacillus subtilis 55.7 32.6 45.3 HisH/E. coli HisC (same gene) IM2/BCA E. coli iluE 63.7 43.6 49.7

[0039] All the clones identified in Table 1 encode polypeptides which have transaminase or aminotransferase activity.

[0040] One means for isolating the nucleic acid molecules encoding the enzymes of the present invention is to probe a gene library with a natural or artificially designed probe using art recognized procedures (see, for example: Current Protocols in Molecular Biology, Ausubel F. M. et al. (EDS.) Green Publishing Company Assoc. and John Wiley Interscience, New York, 1989, 1992). It is appreciated by one skilled in the art that the polynucleotides of SEQ ID NOS:17-24, or fragments thereof (comprising at least 12 contiguous nucleotides), are particularly useful probes. Other particularly useful probes for this purpose are hybridizable fragments of the sequences of SEQ ID NOS:1-9 (i.e., comprising at least 12 contiguous nucleotides).

[0041] With respect to nucleic acid sequences which hybridize to specific nucleic acid sequences disclosed herein, hybridization may be carried out under conditions of reduced stringency, medium stringency or even stringent conditions. As an example of oligonucleotide hybridization, a polymer membrane containing immobilized denatured nucleic acids is first prehybridized for 30 minutes at 45° C. in a solution consisting of 0.9 M NaCl, 50 mM NaH₂PO₄, pH 7.0, 5.0 mM Na₂ EDTA, 0.5% SDS, 10× Denhardt's, and 0.5 mg/mL polyriboadenylic acid. Approximately 2×10⁷ cpm (specific activity 4-9×10⁸ cpm/ug) of ³²P end-labeled oligonucleotide probe are then added to the solution. After 12-16 hours of incubation, the membrane is washed for 30 minutes at room temperature in 1× SET (150 mM NaCl, 20 mM Tris hydrochloride, pH 7.8, 1 mM Na₂EDTA) containing 0.5% SDS, followed by a 30 minute wash in fresh 1× SET at Tm-10° C. (Tm is minus 10° C.) for the oligo-nucleotide probe. The membrane is then exposed to auto-radiographic film for detection of hybridization signals.

[0042] Stringent conditions means hybridization will occur only if there is at least 90% identity, preferably at least 95% identity and most preferably at least 97% identity between the sequences. See J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory (1989) which is hereby incorporated by reference in its entirety.

[0043] As used herein, a first DNA (RNA) sequence is at least 70% and preferably at least 80% identical to another DNA (RNA) sequence if there is at least 70% and preferably at least a 80% or 90% identity, respectively, between the bases of the first sequence and the bases of the another sequence, when properly aligned with each other, for example when aligned by BLASTN.

[0044] The present invention relates to polynucleotides which differ from the reference polynucleotide such that the changes are silent changes, for example the change does not or the changes do not alter the amino acid sequence encoded by the polynucleotide. The present invention also relates to nucleotide changes which result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference polynucleotide. In a preferred aspect of the invention these polypeptides retain the same biological action as the polypeptide encoded by the reference polynucleotide.

[0045] The polynucleotides of this invention were recovered from genomic gene libraries from the organisms listed in Table 1. Gene libraries were generated in the Lambda ZAP II cloning vector (Stratagene Cloning Systems). Mass excisions were performed on these libraries to generate libraries in the pBluescript phagemid. Libraries were generated and excisions were performed according to the protocols/methods hereinafter described.

[0046] The polynucleotides of the present invention may be in the form of RNA or DNA which DNA includes cDNA, genomic DNA, and synthetic DNA. The DNA may be double-stranded or single-stranded, and if single stranded may be the coding strand or non-coding (anti-sense) strand. The coding sequences which encodes the mature enzymes may be identical to the coding sequences shown in FIGS. 1-8 (SEQ ID NOS:17-24) or may be a different coding sequence which coding sequence, as a result of the redundancy or degeneracy of the genetic code, encodes the same mature enzymes as the DNA of FIGS. 1-8 (SEQ ID NOS:17-24).

[0047] The polynucleotide which encodes for the mature enzyme of FIGS. 1-8 (SEQ ID NOS:25-32) may include, but is not limited to: only the coding sequence for the mature enzyme; the coding sequence for the mature enzyme and additional coding sequence such as a leader sequence or a proprotein sequence; the coding sequence for the mature enzyme (and optionally additional coding sequence) and non-coding sequence, such as introns or non-coding sequence 5′ and/or 3′ of the coding sequence for the mature enzyme.

[0048] Thus, the term “polynucleotide encoding an enzyme (protein)” encompasses a polynucleotide which includes only coding sequence for the enzyme as well as a polynucleotide which includes additional coding and/or non-coding sequence.

[0049] The present invention further relates to variants of the hereinabove described polynucleotides which encode for fragments, analogs and derivatives of the enzymes having the deduced amino acid sequences of FIGS. 1-8 (SEQ ID NOS;25-32). The variant of the polynucleotide may be a naturally occurring allelic variant of the polynucleotide or a non-naturally occurring variant of the polynucleotide.

[0050] Thus, the present invention includes polynucleotides encoding the same mature enzymes as shown in FIGS. 1-8 (SEQ ID NOS:17-24) as well as variants of such polyynucleotides which variants encode for a fragment, derivative or analog of the enzymes of FIGS. 1-8 (SEQ ID NOS:17-24). Such nucleotide variants include deletion variants, substitution variants and addition or insertion variants.

[0051] As hereinabove indicated, the polynucleotides may have a coding sequence which is a naturally occurring allelic variant of the coding sequences shown in FIGS. 1-8 (SEQ ID NOS:17-24). As known in the art, an allelic variant is an alternate form of a polynucleotide sequence which may have a substitution, deletion or addition of one or more nucleotides, which does not substantially alter the function of the encoded enzyme. Also, using directed and other evolution strategies, one may make very minor changes in DNA sequence which can result in major changes in function.

[0052] Fragments of the full length gene of the present invention may be used as hybridization probes for a cDNA or a genomic library to isolate the full length DNA and to isolate other DNAs which have a high sequence similarity to the gene or similar biological activity. Probes of this type preferably have at least 10, preferably at least 15, and even more preferably at least 30 bases and may contain, for example, at least 50 or more bases. The probe may also be used to identify a DNA clone corresponding to a full length transcript and a genomic clone or clones that contain the complete gene including regulatory and promotor regions, exons and introns. An example of a screen comprises isolating the coding region of the gene by using the known DNA sequence to synthesize an oligonucleotide probe. Labeled oligonucleotides having a sequence complementary or identical to that of the gene or portion of the gene sequences of the present invention are used to screen a library of genomic DNA to determine which members of the library the probe hybridizes to.

[0053] It is also appreciated that such probes can be and are preferably labeled with an analytically detectable reagent to facilitate identification of the probe. Useful reagents include but are not limited to radioactivity, fluorescent dyes or enzymes capable of catalyzing the formation of a detectable product. The probes are thus useful to isolate complementary copies of DNA from other sources or to screen such sources for related sequences.

[0054] The present invention further relates to polynucleotides which hybridize to the hereinabove-described sequences if there is at least 70%, preferably at least 90%, and more preferably at least 95% identity between the sequences. The present invention particularly relates to polynucleotides which hybridize under stringent conditions to the hereinabove-described polynucleotides. As herein used, the term “stringent conditions” means hybridization will occur only if there is at least 95% and preferably at least 97% identity between the sequences. The polynucleotides which hybridize to the hereinabove described polynucleotides in a preferred embodiment encode enzymes which either retain substantially the same biological function or activity as the mature enzyme encoded by the DNA of FIGS. 1-8 (SEQ ID NOS:17-24).

[0055] Alternatively, the polynucleotide may have at least 15 bases, preferably at least 30 bases, and more preferably at least 50 bases which hybridize to any part of a polynucleotide of the present invention and which has an identity thereto, as hereinabove described, and which may or may not retain activity. For example, such polynucleotides may be employed as probes for the polynucleotides of SEQ ID NOS:17-24, for example, for recovery of the polynucleotide or as a diagnostic probe or as a PCR primer.

[0056] Thus, the present invention is directed to polynucleotides having at least a 70% identity, preferably at least 90% identity and more preferably at least a 95% identity to a polynucleotide which encodes the enzymes of SEQ ID NOS:25-32 as well as fragments thereof, which fragments have at least 15 bases, preferably at least 30 bases and most preferably at least 50 bases, which fragments are at least 90% identical, preferably at least 95% identical and most preferably at least 97% identical under stringent conditions to any portion of a polynucleotide of the present invention.

[0057] The present invention further relates to enzymes which have the deduced amino acid sequences of FIGS. 1-8 (SEQ ID NOS:17-24) as well as fragments, analogs and derivatives of such enzyme.

[0058] The terms “fragment,” “derivative” and “analog” when referring to the enzymes of FIGS. 1-8 (SEQ ID NOS:25-32) means enzymes which retain essentially the same biological function or activity as such enzymes. Thus, an analog includes a proprotein which can be activated by cleavage of the proprotein portion to produce an active mature enzyme.

[0059] The enzymes of the present invention may be a recombinant enzyme, a natural enzyme or a synthetic enzyme, preferably a recombinant enzyme.

[0060] The fragment, derivative or analog of the enzymes of FIGS. 1-8 (SEQ ID NOS:25-32) may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which the mature enzyme is fused with another compound, such as a compound to increase the half-life of the enzyme (for example, polyethylene glycol), or (iv) one in which the additional amino acids are fused to the mature enzyme, such as a leader or secretory sequence or a sequence which is employed for purification of the mature enzyme or a proprotein sequence. Such fragments, derivatives and analogs are deemed to be within the scope of those skilled in the art from the teachings herein.

[0061] The enzymes and polynucleotides of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity.

[0062] The term “isolated” means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring). For example, a naturally-occurring polynucleotide or enzyme present in a living animal is not isolated, but the same polynucleotide or enzyme, separated from some or all of the coexisting materials in the natural system, is isolated. Such polynucleotides could be part of a vector and/or such polynucleotides or enzymes could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment.

[0063] The enzymes of the present invention include the enzymes of SEQ ID NOS:25-32 (in particular the mature enzyme) as well as enzymes which have at least 70% similarity (preferably at least 70% identity) to the enzymes of SEQ ID NOS:25-32 and more preferably at least 90 % similarity (more preferably at least 90% identity) to the enzymes of SEQ ID NOS:25-32 and still more preferably at least 95% similarity (still more preferably at least 95% identity) to the enzymes of SEQ ID NOS:25-32 and also include portions of such enzymes with such portion of the enzyme generally containing at least 30 amino acids and more preferably at least 50 amino acids.

[0064] As known in the art “similarity” between two enzymes is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one enzyme to the sequence of a second enzyme.

[0065] A variant, i.e. a “fragment”, “analog” or “derivative” polypeptide, and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions, fusions and truncations, which may be present in any combination.

[0066] Among preferred variants are those that vary from a reference by conservative amino acid substitutions. Such substitutions are those that substitute a given amino acid in a polypeptide by another amino acid of like characteristics. Typically seen as conservative substitutions are the replacements, one for another, among the aliphatic amino acids Ala, Val, Leu and Ile; interchange of the hydroxyl residues Ser and Thr, exchange of the acidic residues Asp and Glu, substitution between the amide residues Asn and Gln, exchange of the basic residues Lys and Arg and replacements among the aromatic residues Phe, Tyr.

[0067] Most highly preferred are variants which retain the same biological function and activity as the reference polypeptide from which it varies.

[0068] Fragments or portions of the enzymes of the present invention may be employed for producing the corresponding full-length enzyme by peptide synthesis; therefore, the fragments may be employed as intermediates for producing the full-length enzymes. Fragments or portions of the polynucleotides of the present invention may be used to synthesize full-length polynucleotides of the present invention.

[0069] The present invention also relates to vectors which include polynucleotides of the present invention, host cells which are genetically engineered with vectors of the invention and the production of enzymes of the invention by recombinant techniques.

[0070] Host cells are genetically engineered (transduced or transformed or transfected) with the vectors of this invention which may be, for example, a cloning vector such as an expression vector. The vector may be, for example, in the form of a plasmid, a phage, etc. The engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying the genes of the present invention. The culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.

[0071] The polynucleotides of the present invention may be employed for producing enzymes by recombinant techniques. Thus, for example, the polynucleotide may be included in any one of a variety of expression vectors for expressing an enzyme. Such vectors include chromosomal, nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, and pseudorabies. However, any other vector may be used as long as it is replicable and viable in the host.

[0072] The appropriate DNA sequence may be inserted into the vector by a variety of procedures. In general, the DNA sequence is inserted into an appropriate restriction endonuclease site(s) by procedures known in the art. Such procedures and others are deemed to be within the scope of those skilled in the art.

[0073] The DNA sequence in the expression vector is operatively linked to an appropriate expression control sequence(s) (promoter) to direct mRNA synthesis. As representative examples of such promoters, there may be mentioned: LTR or SV40 promoter, the E. coli. Zac or tip, the phage lambda P_(L) promoter and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses. The expression vector also contains a ribosome binding site for translation initiation and a transcription terminator. The vector may also include appropriate sequences for amplifying expression.

[0074] In addition, the expression vectors preferably contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in E. coli.

[0075] The vector containing the appropriate DNA sequence as hereinabove described, as well as an appropriate promoter or control sequence, may be employed to transform an appropriate host to permit the host to express the protein.

[0076] As representative examples of appropriate hosts, there may be mentioned: bacterial cells, such as E. coli, Streptomyces, Bacillus subtilis; fungal cells, such as yeast; insect cells such as Drosophila S2 and Spodoptera SJ9; animal cells such as CHO, COS or Bowes melanoma; adenoviruses; plant cells, etc. The selection of an appropriate host is deemed to be within the scope of those skilled in the art from the teachings herein.

[0077] More particularly, the present invention also includes recombinant constructs comprising one or more of the sequences as broadly described above. The constructs comprise a vector, such as a plasmid or viral vector, into which a sequence of the invention has been inserted, in a forward or reverse orientation. In a preferred aspect of this embodiment, the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence. Large numbers of suitable vectors and promoters are known to those of skill in the art, and are commercially available. The following vectors are provided by way of example; Bacterial: pQE70, pQE60, pQE-9 (Qiagen), pBluescript II KS, ptrc99a, pKK223-3, pDR540, pRIT2T (Pharmacia); Eukaryotic: pXT1, pSG5 (Stratagene) pSVK3, pBPV, pMSG, pSVL SV40 (Pharmacia). However, any other plasmid or vector may be used as long as they are replicable and viable in the host.

[0078] Promoter regions can be selected from any desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers. Two appropriate vectors are pKK232-8 and pCM7. Particular named bacterial promoters include lac, lacZ, T3, T7, gpt, lambda P_(R), P_(L) and trp. Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.

[0079] In a further embodiment, the present invention relates to host cells containing the above-described constructs. The host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-Dextran mediated transfection, or electroporation (Davis, L., Dibner, M., Battey, I., Basic Methods in Molecular Biology, (1986)).

[0080] The constructs in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence. Alternatively, the enzymes of the invention can be synthetically produced by conventional peptide synthesizers.

[0081] Mature proteins can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention. Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), the disclosure of which is hereby incorporated by reference.

[0082] Transcription of the DNA encoding the enzymes of the present invention by higher eukaryotes is increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act on a promoter to increase its transcription. Examples include the SV40 enhancer on the late side of the replication origin bp 100 to 270, a cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

[0083] Generally, recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiae TRP1 gene, and a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence. Such promoters can be derived from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), α-factor, acid phosphatase, or heat shock proteins, among others. The heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated enzyme. Optionally, the heterologous sequence can encode a fusion enzyme including an N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product.

[0084] Useful expression vectors for bacterial use are constructed by inserting a structural DNA sequence encoding a desired protein together with suitable translation initiation and termination signals in operable reading phase with a functional promoter. The vector will comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to, if desirable, provide amplification within the host. Suitable prokaryotic hosts for transformation include E. coli, Bacillus subtilis, Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus, although others may also be employed as a matter of choice.

[0085] As a representative but nonlimiting example, useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC 37017). Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and pGEM1 (Promega Biotec, Madison, Wis., USA). These pBR322 “backbone” sections are combined with an appropriate promoter and the structural sequence to be expressed.

[0086] Following transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter is induced by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period.

[0087] Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.

[0088] Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents, such methods are well known to those skilled in the art.

[0089] Various mammalian cell culture systems can also be employed to express recombinant protein. Examples of mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell, 23:175 (1981), and other cell lines capable of expressing a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines. Mammalian expression vectors will comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5′ flanking nontranscribed sequences. DNA sequences derived from the SV40 splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements.

[0090] The enzyme can be recovered and purified from recombinant cell cultures by methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Protein refolding steps can be used, as necessary, in completing configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps.

[0091] The enzymes of the present invention may be a naturally purified product, or a product of chemical synthetic procedures, or produced by recombinant techniques from a prokaryotic or eukaryotic host (for example, by bacterial, yeast, higher plant, insect and mammalian cells in culture). Depending upon the host employed in a recombinant production procedure, the enzymes of the present invention may be glycosylated or may be non-glycosylated. Enzymes of the invention may or may not also include an initial methionine amino acid residue.

[0092] Transaminases are a group of key enzymes in the metabolism of amino acids and amino sugars and are found in all organisms from microbes to mammals. In the transamination reaction, an amino group is transferred from an amino acid to an α-keto acid. Pyridoxal phosphate is required as a co-factor to mediate the transfer of the amino group without liberation of ammonia.

[0093] Amino acids currently have applications as additives to aminal feed, human nutritional supplements, components in infusion solutions, and synthetic intermediates for manufacture of pharmaceuticals and agricultural products. For example, L-glutamic acid is best known as a flavor enhancer for human food. L-lysine and L-methionine are large volume additives to animal feed and human supplements. L-tryptophan and L-threonine have similar potential applications. L-phenylalanine and L-aspartic acid have very important market potential as key components in the manufacture of the low-calorie sweetener aspartame, and other promising low-calorie sweeteners have compositions containing certain amino acids as well. Infusion solutions require a large range of amino acids including those essential ones in human diets.

[0094] Transaminases are highly stereoselective, and most use L-amino acids as substrates. Using the approach disclosed in a commonly assigned, copending provisional application Serial No. 60/008,316, filed on Dec. 7, 1995 and entitled “Combinatorial Enzyme Development,” the disclosure of which is incorporated herein by reference in its entirety, one can convert the transaminases of the invention to use D-amino acids as substrates. Such conversion makes possible a broader array of transaminase applications. For instance, D-valine can be used in the manufacture of synthetic pyrethroids. D-phenylglycine and its derivatives can be useful as components of β-lactam antibiotics. Further, the thermostable transaminases have superior stability at higher temperatures and in organic solvents. Thus, they are better suited to utilize either L- and/or D-amino acids for production of optically pure chiral compounds used in pharmaceutical, agricultural, and other chemical manufactures.

[0095] There are a number of reasons to employ transaminases in industrial-scale production of amino acids and their derivatives.

[0096] 1) Transaminases can catalyze stereoselective synthesis of D- or L-amino acids from their corresponding α-keto acids. Therefore no L- or D-isomers are produced, and no resolution is required.

[0097] 2) Tramminases have uniformly high catalytic rates, capable of converting up to 400 μmoles of substrates per minute per mg enzyme.

[0098] 3) Many required α-keto acids can be conveniently prepared by chemical synthesis at low cost.

[0099] 4) The capital investment for an immobilized enzyme process using transaminases is much lower than for a large scale fermentation process, and productivity of the bioreactor is often an order of magnitude higher.

[0100] 5) The technology is generally applicable to a broad range of D- or L-amino acids because transaminases exist with varying specificities. Such broad scope allows a number of different L- or D-amino acids to be produced with the same equipment and often the same biocatalyst.

[0101] Antibodies generated against the enzymes corresponding to a sequence of the present invention can be obtained by direct injection of the enzymes into an animal or by administering the enzymes to an animal, preferably a nonhuman. The antibody so obtained will then bind the enzymes itself. In this manner, even a sequence encoding only a fragment of the enzymes can be used to generate antibodies binding the whole native enzymes. Such antibodies can then be used to isolate the enzyme from cells expressing that enzyme.

[0102] For preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique (Kohler and Milstein, Nature, 256:495-497, 1975), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., Immunology Today 4:72, 1983), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96, 1985).

[0103] Techniques described for the production of single chain antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce single chain antibodies to immunogenic enzyme products of this invention. Also, transgenic mice may be used to express humanized antibodies to immunogenic enzyme products of this invention.

[0104] Antibodies generated against an enzyme of the present invention may be used in screening for similar enzymes from other organisms and samples. Such screening techniques are known in the art, for example, one such screening assay is described in Sambrook and Maniatis, Molecular Cloning: A Laboratory Manual (2d Ed.), vol. 2:Section 8.49, Cold Spring Harbor Laboratory, 1989, which is hereby incorporated by reference in its entirety.

[0105] The present invention will be further described with reference to the following examples; however, it is to be understood that the present invention is not limited to such examples. All parts or amounts, unless otherwise specified, are by weight.

[0106] In order to facilitate understanding of the following examples certain frequently occurring methods and/or terms will be described.

[0107] “Plasmids” are designated by a lower case “p” preceded and/or followed by capital letters and/or numbers. The starting plasmids herein are either commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids in accord with published procedures. In addition, equivalent plasmids to those described are known in the art and will be apparent to the ordinarily skilled artisan.

[0108] “Digestion” of DNA refers to catalytic cleavage of the DNA with a restriction enzyme that acts only at certain sequences in the DNA. The various restriction enzymes used herein are commercially available and their reaction conditions, cofactors and other requirements were used as would be known to the ordinarily skilled artisan. For analytical purposes, typically 1 μg of plasmid or DNA fragment is used with about 2 units of enzyme in about 20 μl of buffer solution. For the purpose of isolating DNA fragments for plasmid construction, typically 5 to 50 μg of DNA are digested with 20 to 250 units of enzyme in a larger volume. Appropriate buffers and substrate amounts for particular restriction enzymes are specified by the manufacturer. Incubation times of about 1 hour at 37° C. are ordinarily used, but may vary in accordance with the supplier's instructions. After digestion the reaction is electrophoresed directly on a polyacrylamide gel to isolate the desired fragment.

[0109] Size separation of the cleaved fragments is performed using 8 percent polyacrylamide gel described by Goeddel et al., Nucleic Acids Res., 8:4057 (1980).

[0110] “Oligonucleotides” refers to either a single stranded polydeoxynucleotide or two complementary polydeoxynucleotide strands which may be chemically synthesized. Such synthetic oligonucleotides have no 5′ phosphate and thus will not ligate to another oligonucleotide without adding a phosphate with an ATP in the presence of a kinase. A synthetic oligonucleotide will ligate to a fragment that has not been dephosphorylated.

[0111] “Ligation” refers to the process of forming phosphodiester bonds between two double stranded nucleic acid fragments (Maniatis, T., et al., Id., p. 146). Unless otherwise provided, ligation may be accomplished using known buffers and conditions with 10 units of T4 DNA ligase (“ligase”) per 0.5 μg of approximately equimolar amounts of the DNA fragments to be ligated.

[0112] Unless otherwise stated, transformation was performed as described in Sambrook and Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, 1989.

EXAMPLE 1 Bacterial Expression and Purification of Transaminases and Aminotransferases

[0113] DNA encoding the enzymes of the present invention, SEQ ID NOS:25 through 32, were initially amplified from a pBluescript vector containing the DNA by the PCR technique using the primers noted herein. The amplified sequences were then inserted into the respective PQE vector listed beneath the primer sequences, and the enzyme was expressed according to the protocols set forth herein. The genomic DNA has also been used as a template for the PCR amplification, i.e., once a positive clone has been identified and primer sequences determined using the cDNA, it was then possible to return to the genomic DNA and directly amplify the desired sequence(s) there. The 5′ and 3′ primer sequences and the vector for the respective genes are as follows: Aquifex  Aspartate Transaminase A aspa501 5′ CCGAGAATTCATTAAAGAGGAGAAATTAACTATGATGAAGACCCTATGGAC (SEQ. ID NO:1) aspa301 3′ CGAAGATCTTTAGCACTTCTCTCAGGTTC (SEQ. ID NO:2) vector: pQET1 Aquifex  Aspartate Aminotransferase B aspb501 5′ CCGAGAATTCATTAAAGAGGAGAAATTAACTATGGACAGGCTTGAAAAAGTA (SEQ ID NO:3) aspb301 3′ CGGAAGATCTTCAGCTAAGCTTCTCTAAGAA (SEQ ID NO:4) vector: pQET1 Aquifex  Adenosyl-8-amino-7-oxononanoate Aminotransferase ameth501 5′ CCGACAATTGATAAGAGGAGAAATTAACTATGTGGGAATTAGACCCTAAA (SEQ ID NO:5) ameth301 3′ CGGAGGATCCCTACACCTCTTTTTCAAGCT (SEQ ID NO:6) vector: pQET12 Aquifex  Acetylornithine Aminotransferase aorn 501 5′ CCGACAATTGATTAAAGAGGAGAAATTAACTATGACATACTTAATGAACAAT (SEQ ID NO:7) aorn 301 3′ CGGAAGATCTTTATGAGAAGTCCCTTTCAAG (SEQ ID NO:8) vector: pQET12 Ammonifex  desgensii Aspartate Aminotransferase adasp 501 5′ CCGAGAATTCATTAAAGAGGAGAAATTAACTATGCGGAAACTGGCCGAGCGG (SEQ ID NO:9) adasp 301 3′ CGGAGGATCCTTAAAGTGCCGCTTCGATCAA (SEQ ID NO:10) vector: pQET12 Aquifex Glucosamine:Fructose-6-phosphate Aminotransferase glut 501 5′ CCGACAATTGATTAAAGAGGAGAAATTAACTATGTGCGGGATAGTCGGATAC (SEQ ID NO:11) glut 301 3′ CGGAAGATCTTTATTCCACCGTGACCGTTTT (SEQ ID NO:12) vector: pQET1 Aquifex  Histadine-phosphate Aminotransferase his 501 5′ CCGACAATTGATTAAAGAGGAGAAATTAACTATGATACCCCAGAGGATTAAG (SEQ ID NO:13) his 301 3′ CGGAAGATCTTTAAAGAGAGCTTGAAAGGGA (SEQ ID NO:14) vector: pQET1 Pyrobacullum aerophilum  Branched Chain Aminotransferase bcat 501 5′ CCGAGAATTCATTAAAGAGGAGAAATTAACTATGAAGCCGTACGCTAAATAT (SEQ ID NO:15) bcat 301 3′ CGGAAGATCTCTAATACACAGGAGTGATCCA (SEQ ID NO:16) vector: pQET1

[0114] The restriction enzyme sites indicated correspond to the restriction enzyme sites on the bacterial expression vector indicated for the respective gene (Qiagen, Inc. Chatsworth, Calif.). The pQE vector encodes antibiotic resistance (Amp^(r)), a bacterial origin of replication (ori), an IPTG-regulatable promoter operator (P/O), a ribosome binding site (RBS), a 6-His tag and restriction enzyme sites.

[0115] The pQE vector was digested with the restriction enzymes indicated. The amplified sequences were ligated into the respective pQE vector and inserted in frame with the sequence encoding for the RBS. The ligation mixture was then used to transform the E. coli strain M15/pREP4 (Qiagen, Inc.) by electroporation. M15/pREP4 contains multiple copies of the plasmid pREP4, which expresses the lacI repressor and also confers kanamycin resistance (Kan^(r)). Transformants were identified by their ability to grow on LB plates and ampicillin/kanamycin resistant colonies were selected. Plasmid DNA was isolated and confirmed by restriction analysis. Clones containing the desired constructs were grown overnight (O/N) in liquid culture in LB media supplemented with both Amp (100 ug/ml) and Kan (25 ug/ml). The O/N culture was used to inoculate a large culture at a ratio of 1:100 to 1:250. The cells were grown to an optical density 600 (O.D.⁶⁰⁰) of between 0.4 and 0.6. IPTG (“Isopropyl-B-D-thiogalacto pyranoside”) was then added to a fmal concentration of 1 mM. IPTG induces by inactivating the lacI repressor, clearing the P/O leading to increased gene expression. Cells were grown an extra 3 to 4 hours. Cells were then harvested by centrifugation.

[0116] The primer sequences set out above may also be employed to isolate the target gene from the deposited material by hybridization techniques described above.

EXAMPLE 2 Isolation of a Selected Clone from the Deposited Genomic Clones

[0117] The two oligonucleotide primers corresponding to the gene of interest are used to amplify the gene from the deposited material. A polymerase chain reaction is carried out in 25 μl of reaction mixture with 0.1 μg of the DNA of the gene of interest. The reaction mixture is 1.5-5 mM MgCl₂, 0.01 % (w/v) gelatin, 20 μM each of dATP, dCTP, dGTP, dTTP, 25 pmol of each primer and 1.25 Unit of Taq polymerase. Thirty cycles of PCR (denaturation at 94° C. for 1 min; annealing at 55° C. for 1 min; elongation at 72° C. for 1 min) are performed with the Perkin-Elmer Cetus 9600 thermal cycler. The amplified product is analyzed by agarose gel electrophoresis and the DNA band with expected molecular weight is excised and purified. The PCR product is verified to be the gene of interest by subcloning and sequencing the DNA product.

[0118] Numerous modifications and variations of the present invention are possible in light of the above teachings and, therefore, within the scope of the appended claims, the invention may be practiced otherwise than as particularly described.

1 32 52 NUCLEOTIDES NUCLEIC ACID SINGLE LINEAR cDNA 1 CCGAGAATTC ATTAAAGAGG AGAAATTAAC TATGATTGAA GACCCTATGG AC 52 31 NUCLEOTIDES NUCLEIC ACID SINGLE LINEAR cDNA 2 CGGAAGATCT TTAAGCACTT CTCTCAGGTT C 31 52 NUCLEOTIDES NUCLEIC ACID SINGLE LINEAR cDNA 3 CCGAGAATTC ATTAAAGAGG AGAAATTAAC TATGGACAGG CTTGAAAAAG TA 52 31 NUCLEOTIDES NUCLEIC ACID SINGLE LINEAR cDNA 4 CGGAAGATCT TCAGCTAAGC TTCTCTAAGA A 31 52 NUCLEOTIDES NUCLEIC ACID SINGLE LINEAR cDNA 5 CCGACAATTG ATTAAAGAGG AGAAATTAAC TATGTGGGAA TTAGACCCTA AA 52 31 NUCLEOTIDES NUCLEIC ACID SINGLE LINEAR cDNA 6 CGGAGGATCC CTACACCTGT TTTTCAAGCT C 31 52 NUCLEOTIDES NUCLEIC ACID SINGLE LINEAR cDNA 7 CCGACAATTG ATTAAAGAGG AGAAATTAAC TATGACATAC TTAATGAACA AT 52 31 NUCLEOTIDES NUCLEIC ACID SINGLE LINEAR cDNA 8 CGGAAGATCT TTATGAGAAG TCCCTTTCAA G 31 52 NUCLEOTIDES NUCLEIC ACID SINGLE LINEAR cDNA 9 CCGAGAATTC ATTAAAGAGG AGAAATTAAC TATGCGGAAA CTGGCCGAGC GG 52 31 NUCLEOTIDES NUCLEIC ACID SINGLE LINEAR cDNA 10 CGGAGGATCC TTAAAGTGCC GCTTCGATCA A 31 52 NUCLEOTIDES NUCLEIC ACID SINGLE LINEAR cDNA 11 CCGACAATTG ATTAAAGAGG AGAAATTAAC TATGTGCGGG ATAGTCGGAT AC 52 31 NUCLEOTIDES NUCLEIC ACID SINGLE LINEAR cDNA 12 CGGAAGATCT TTATTCCACC GTGACCGTTT T 31 52 NUCLEOTIDES NUCLEIC ACID SINGLE LINEAR cDNA 13 CCGACAATTG ATTAAAGAGG AGAAATTAAC TATGATACCC CAGAGGATTA AG 52 31 NUCLEOTIDES NUCLEIC ACID SINGLE LINEAR cDNA 14 CGGAAGATCT TTAAAGAGAG CTTGAAAGGG A 31 52 NUCLEOTIDES NUCLEIC ACID SINGLE LINEAR cDNA 15 CCGAGAATTC ATTAAAGAGG AGAAATTAAC TATGAAGCCG TACGCTAAAT AT 52 31 NUCLEOTIDES NUCLEIC ACID SINGLE LINEAR cDNA 16 CGGAAGATCT CTAATACACA GGAGTGATCC A 31 1245 NUCLEOTIDES NUCLEIC ACID SINGLE LINEAR GENOMIC DNA 17 ATG ATT GAA GAC CCT ATG GAC TGG GCT TTT CCG AGG ATA AAG AGA CTG 48 Met Ile Glu Asp Pro Met Asp Trp Ala Phe Pro Arg Ile Lys Arg Leu 5 10 15 CCT CAG TAT GTC TTC TCT CTC GTT AAC GAA CTC AAG TAC AAG CTA AGG 96 Pro Gln Tyr Val Phe Ser Leu Val Asn Glu Leu Lys Tyr Lys Leu Arg 20 25 30 CGT GAA GGC GAA GAT GTA GTG GAT CTT GGT ATG GGC AAT CCT AAC ATG 144 Arg Glu Gly Glu Asp Val Val Asp Leu Gly Met Gly Asn Pro Asn Met 35 40 45 CCT CCA GCA AAG CAC ATA ATA GAT AAA CTC TGC GAA GTG GCT CAA AAG 192 Pro Pro Ala Lys His Ile Ile Asp Lys Leu Cys Glu Val Ala Gln Lys 50 55 60 CCG AAC GTT CAC GGA TAT TCT GCG TCA AGG GGC ATA CCA AGA CTG AGA 240 Pro Asn Val His Gly Tyr Ser Ala Ser Arg Gly Ile Pro Arg Leu Arg 65 70 75 80 AAG GCT ATA TGT AAC TTC TAC GAA GAA AGG TAC GGA GTG AAA CTC GAC 288 Lys Ala Ile Cys Asn Phe Tyr Glu Glu Arg Tyr Gly Val Lys Leu Asp 85 90 95 CCT GAG AGG GAG GCT ATA CTA ACA ATC GGT GCA AAG GAA GGG TAT TCT 336 Pro Glu Arg Glu Ala Ile Leu Thr Ile Gly Ala Lys Glu Gly Tyr Ser 100 105 110 CAT TTG ATG CTT GCG ATG ATA TCT CCG GGT GAT ACG GTA ATA GTT CCT 384 His Leu Met Leu Ala Met Ile Ser Pro Gly Asp Thr Val Ile Val Pro 115 120 125 AAT CCC ACC TAT CCT ATT CAC TAT TAC GCT CCC ATA ATT GCA GGA GGG 432 Asn Pro Thr Tyr Pro Ile His Tyr Tyr Ala Pro Ile Ile Ala Gly Gly 130 135 140 GAA GTT CAC TCA ATA CCC CTT AAC TTC TCG GAC GAT CAA GAT CAT CAG 480 Glu Val His Ser Ile Pro Leu Asn Phe Ser Asp Asp Gln Asp His Gln 145 150 155 160 GAA GAG TTT TTA AGG AGG CTT TAC GAG ATA GTA AAA ACC GCG ATG CCA 528 Glu Glu Phe Leu Arg Arg Leu Tyr Glu Ile Val Lys Thr Ala Met Pro 165 170 175 AAA CCC AAG GCT GTC GTC ATA AGC TTT CCT CAC AAT CCA ACG ACC ATA 576 Lys Pro Lys Ala Val Val Ile Ser Phe Pro His Asn Pro Thr Thr Ile 180 185 190 ACG GTA GAA AAG GAC TTT TTT AAA GAA ATA GTT AAG TTT GCA AAG GAA 624 Thr Val Glu Lys Asp Phe Phe Lys Glu Ile Val Lys Phe Ala Lys Glu 195 200 205 CAC GGT CTC TGG ATA ATA CAC GAT TTT GCG TAT GCG GAT ATA GCC TTT 672 His Gly Leu Trp Ile Ile His Asp Phe Ala Tyr Ala Asp Ile Ala Phe 210 215 220 GAC GGT TAC AAG CCC CCC TCA ATA CTC GAA ATA GAA GGT GCT AAA GAC 720 Asp Gly Tyr Lys Pro Pro Ser Ile Leu Glu Ile Glu Gly Ala Lys Asp 225 230 235 240 GTT GCG GTT GAG CTC TAC TCC ATG TCA AAG GGC TTT TCA ATG GCG GGC 768 Val Ala Val Glu Leu Tyr Ser Met Ser Lys Gly Phe Ser Met Ala Gly 245 250 255 TGG AGG GTA GCC TTT GTC GTT GGA AAC GAA ATA CTC ATA AAA AAC CTT 816 Trp Arg Val Ala Phe Val Val Gly Asn Glu Ile Leu Ile Lys Asn Leu 260 265 270 GCA CAC CTC AAA AGC TAC TTG GAT TAC GGT ATA TTT ACT CCC ATA CAG 864 Ala His Leu Lys Ser Tyr Leu Asp Tyr Gly Ile Phe Thr Pro Ile Gln 275 280 285 GTG GCC TCT ATT ATC GCA TTA GAG AGC CCC TAC GAA ATC GTG GAA AAA 912 Val Ala Ser Ile Ile Ala Leu Glu Ser Pro Tyr Glu Ile Val Glu Lys 290 295 300 ACC GCA AAG GTT TAC CAA AAA AGA AGA GAC GTT CTG GTG GAA GGG TTA 960 Thr Ala Lys Val Tyr Gln Lys Arg Arg Asp Val Leu Val Glu Gly Leu 305 310 315 320 AAC AGG CTC GGC TGG AAA GTA AAA AAA CCT AAG GCT ACC ATG TTC GTC 1008 Asn Arg Leu Gly Trp Lys Val Lys Lys Pro Lys Ala Thr Met Phe Val 325 330 335 TGG GCA AAG ATT CCC GAA TGG ATA AAT ATG AAC TCT CTG GAC TTT TCC 1056 Trp Ala Lys Ile Pro Glu Trp Ile Asn Met Asn Ser Leu Asp Phe Ser 340 345 350 TTG TTC CTC CTA AAA GAG GCG AAG GTT GCG GTA TCC CCG GGT GTG GGC 1104 Leu Phe Leu Leu Lys Glu Ala Lys Val Ala Val Ser Pro Gly Val Gly 355 360 365 TTT GGT CAG TAC GGA GAG GGG TAC GTA AGG TTT GCA CTT GTA GAA AAT 1152 Phe Gly Gln Tyr Gly Glu Gly Tyr Val Arg Phe Ala Leu Val Glu Asn 370 375 380 GAA CAC AGG ATC AGA CAG GCT ATA AGG GGA ATA AGG AAA GCC TTC AGA 1200 Glu His Arg Ile Arg Gln Ala Ile Arg Gly Ile Arg Lys Ala Phe Arg 385 390 395 400 AAA CTC CAG AAG GAG AGG AAA CTT GAA CCT GAG AGA AGT GCT TAA 1245 Lys Leu Gln Lys Glu Arg Lys Leu Glu Pro Glu Arg Ser Ala End 405 410 414 1122 NUCLEOTIDES NUCLEIC ACID SINGLE LINEAR GENOMIC DNA 18 ATG GAC AGG CTT GAA AAA GTA TCA CCC TTC ATA GTA ATG GAT ATC CTA 48 Met Asp Arg Leu Glu Lys Val Ser Pro Phe Ile Val Met Asp Ile Leu 5 10 15 GCT CAG GCC CAG AAG TAC GAA GAC GTA GTA CAC ATG GAG ATA GGA GAG 96 Ala Gln Ala Gln Lys Tyr Glu Asp Val Val His Met Glu Ile Gly Glu 20 25 30 CCC GAT TTA GAA CCG TCT CCC AAG GTA ATG GAA GCT CTG GAA CGT GCG 144 Pro Asp Leu Glu Pro Ser Pro Lys Val Met Glu Ala Leu Glu Arg Ala 35 40 45 GTG AAG GAA AAG ACG TTC TTC TAC ACC CCT GCT CTG GGA CTC TGG GAA 192 Val Lys Glu Lys Thr Phe Phe Tyr Thr Pro Ala Leu Gly Leu Trp Glu 50 55 60 CTC AGG GAA AGG ATA TCG GAG TTT TAC AGG AAA AAG TAC AGC GTT GAA 240 Leu Arg Glu Arg Ile Ser Glu Phe Tyr Arg Lys Lys Tyr Ser Val Glu 65 70 75 80 GTT TCT CCA GAG AGA GTC ATC GTA ACT ACC GGA ACT TCG GGA GCG TTT 288 Val Ser Pro Glu Arg Val Ile Val Thr Thr Gly Thr Ser Gly Ala Phe 85 90 95 CTC GTA GCC TAC GCC GTA ACA CTA AAT GCG GGA GAG AAG ATA ATC CTC 336 Leu Val Ala Tyr Ala Val Thr Leu Asn Ala Gly Glu Lys Ile Ile Leu 100 105 110 CCA GAC CCC TCT TAC CCC TGT TAC AAA AAC TTT GCC TAC CTC TTA GAC 384 Pro Asp Pro Ser Tyr Pro Cys Tyr Lys Asn Phe Ala Tyr Leu Leu Asp 115 120 125 GCT CAG CCG GTT TTC GTA AAC GTT GAC AAG GAA ACG AAT TAC GAA GTA 432 Ala Gln Pro Val Phe Val Asn Val Asp Lys Glu Thr Asn Tyr Glu Val 130 135 140 AGG AAA GAG ATG ATA GAA GAC ATT GAT GCG AAA GCC CTT CAC ATT TCC 480 Arg Lys Glu Met Ile Glu Asp Ile Asp Ala Lys Ala Leu His Ile Ser 145 150 155 160 TCG CCT CAA AAC CCT ACG GGC ACA CTC TAC TCA CCT GAA ACC CTG AAG 528 Ser Pro Gln Asn Pro Thr Gly Thr Leu Tyr Ser Pro Glu Thr Leu Lys 165 170 175 GAA CTT GCG GAG TAC TGC GAA GAG AAG GGT ATG TAC TTC ATA TCC GAC 576 Glu Leu Ala Glu Tyr Cys Glu Glu Lys Gly Met Tyr Phe Ile Ser Asp 180 185 190 GAG ATT TAC CAC GGA CTC GTT TAC GAA GGT AGG GAG CAC ACA GCA CTT 624 Glu Ile Tyr His Gly Leu Val Tyr Glu Gly Arg Glu His Thr Ala Leu 195 200 205 GAG TTC TCT GAC AGG GCT ATT GTC ATA AAC GGG TTT TCT AAG TAC TTC 672 Glu Phe Ser Asp Arg Ala Ile Val Ile Asn Gly Phe Ser Lys Tyr Phe 210 215 220 TGT ATG CCA GGT TTC AGG ATA GGG TGG ATG ATA GTT CCG GAA GAA CTC 720 Cys Met Pro Gly Phe Arg Ile Gly Trp Met Ile Val Pro Glu Glu Leu 225 230 235 240 GTG AGA AAG GCG GAA ATA GTA ATT CAG AAC GTA TTT ATA TCT GCC CCG 768 Val Arg Lys Ala Glu Ile Val Ile Gln Asn Val Phe Ile Ser Ala Pro 245 250 255 ACG CTC AGT CAG TAC GCC GCC CTT GAG GCT TTT GAT TAC GAG TAT TTG 816 Thr Leu Ser Gln Tyr Ala Ala Leu Glu Ala Phe Asp Tyr Glu Tyr Leu 260 265 270 GAG AAG GTA AGA AAA ACC TTT GAA GAG AGG AGG AAC TTC CTT TAT GGG 864 Glu Lys Val Arg Lys Thr Phe Glu Glu Arg Arg Asn Phe Leu Tyr Gly 275 280 285 GAA CTG AAA AAA CTC TTC AAG ATA GAC GCG AAA CCT CAG GGA GCT TTT 912 Glu Leu Lys Lys Leu Phe Lys Ile Asp Ala Lys Pro Gln Gly Ala Phe 290 295 300 TAC GTA TGG GCA AAC ATA AGT GAT TAC TCC ACA GAT AGC TAC GAA TTT 960 Tyr Val Trp Ala Asn Ile Ser Asp Tyr Ser Thr Asp Ser Tyr Glu Phe 305 310 315 320 GCT TTA AAA CTT TTA AGG GAG GCG AGG GTG GCG GTA ACG CCC GGG GTG 1008 Ala Leu Lys Leu Leu Arg Glu Ala Arg Val Ala Val Thr Pro Gly Val 325 330 335 GAC TTT GGA AAA AAC AAA ACG AAG GAG TAT ATA AGG TTT GCT TAT ACG 1056 Asp Phe Gly Lys Asn Lys Thr Lys Glu Tyr Ile Arg Phe Ala Tyr Thr 340 345 350 AGA AAG ATA GAA GAA CTT AAG GAG GGC GTT GAA AGG ATA AAG AAG TTC 1104 Arg Lys Ile Glu Glu Leu Lys Glu Gly Val Glu Arg Ile Lys Lys Phe 355 360 365 TTA GAG AAG CTT AGC TGA 1122 Leu Glu Lys Leu Ser End 370 1362 NUCLEOTIDES NUCLEIC ACID SINGLE LINEAR GENOMIC DNA 19 ATG TGG GAA TTA GAC CCT AAA ACG CTC GAA AAG TGG GAC AAG GAG TAC 48 Met Trp Glu Leu Asp Pro Lys Thr Leu Glu Lys Trp Asp Lys Glu Tyr 5 10 15 TTC TGG CAT CCA TTT ACC CAG ATG AAA GTC TAC AGA GAA GAA GAA AAC 96 Phe Trp His Pro Phe Thr Gln Met Lys Val Tyr Arg Glu Glu Glu Asn 20 25 30 CTG ATA TTT GAA CGC GGA GAA GGC GTT TAC CTG TGG GAC ATA TAC GGC 144 Leu Ile Phe Glu Arg Gly Glu Gly Val Tyr Leu Trp Asp Ile Tyr Gly 35 40 45 AGG AAG TAT ATA GAT GCC ATA TCT TCC CTC TGG TGC AAC GTC CAC GGA 192 Arg Lys Tyr Ile Asp Ala Ile Ser Ser Leu Trp Cys Asn Val His Gly 50 55 60 CAT AAC CAC CCT AAA CTG AAC AAC GCA GTT ATG AAA CAG CTC TGT AAG 240 His Asn His Pro Lys Leu Asn Asn Ala Val Met Lys Gln Leu Cys Lys 65 70 75 80 GTA GCT CAC ACA ACT ACT CTG GGA AGT TCC AAC GTT CCC GCC ATA CTC 288 Val Ala His Thr Thr Thr Leu Gly Ser Ser Asn Val Pro Ala Ile Leu 85 90 95 CTT GCA AAG AAG CTT GTA GAA ATT TCT CCT GAA GGA TTA AAC AAG GTC 336 Leu Ala Lys Lys Leu Val Glu Ile Ser Pro Glu Gly Leu Asn Lys Val 100 105 110 TTT TAC TCC GAA GAC GGT GCG GAA GCA GTA GAG ATA GCG ATA AAG ATG 384 Phe Tyr Ser Glu Asp Gly Ala Glu Ala Val Glu Ile Ala Ile Lys Met 115 120 125 GCT TAT CAC TAC TGG AAG AAC AAG GGA GTT AAA GGG AAA AAC GTT TTC 432 Ala Tyr His Tyr Trp Lys Asn Lys Gly Val Lys Gly Lys Asn Val Phe 130 135 140 ATA ACG CTT TCC GAA GCC TAC CAC GGG GAT ACT GTA GGA GCG GTT AGC 480 Ile Thr Leu Ser Glu Ala Tyr His Gly Asp Thr Val Gly Ala Val Ser 145 150 155 160 GTA GGG GGT ATA GAA CTC TTC CAC GGA ACT TAT AAA GAT CTC CTT TTC 528 Val Gly Gly Ile Glu Leu Phe His Gly Thr Tyr Lys Asp Leu Leu Phe 165 170 175 AAG ACT ATA AAA CTC CCA TCT CCT TAC CTG TAC TGC AAG GAA AAG TAC 576 Lys Thr Ile Lys Leu Pro Ser Pro Tyr Leu Tyr Cys Lys Glu Lys Tyr 180 185 190 GGG GAA CTC TGC CCT GAG TGC ACG GCA GAT TTA TTA AAA CAA CTG GAA 624 Gly Glu Leu Cys Pro Glu Cys Thr Ala Asp Leu Leu Lys Gln Leu Glu 195 200 205 GAT ATC CTG AAG TCG CGG GAA GAT ATC GTT GCG GTC ATT ATG GAA GCG 672 Asp Ile Leu Lys Ser Arg Glu Asp Ile Val Ala Val Ile Met Glu Ala 210 215 220 GGA ATT CAG GCA GCC GCG GGA ATG CTC CCC TTC CCT CCG GGA TTT TTG 720 Gly Ile Gln Ala Ala Ala Gly Met Leu Pro Phe Pro Pro Gly Phe Leu 225 230 235 240 AAA GGC GTA AGG GAG CTT ACG AAG AAA TAC GAC ACT TTA ATG ATA GTT 768 Lys Gly Val Arg Glu Leu Thr Lys Lys Tyr Asp Thr Leu Met Ile Val 245 250 255 GAC GAG GTT GCC ACG GGA TTT GGC AGG ACG GGA ACG ATG TTT TAC TGT 816 Asp Glu Val Ala Thr Gly Phe Gly Arg Thr Gly Thr Met Phe Tyr Cys 260 265 270 GAG CAG GAA GGA GTC AGT CCG GAC TTT ATG TGT CTA GGT AAG GGT ATA 864 Glu Gln Glu Gly Val Ser Pro Asp Phe Met Cys Leu Gly Lys Gly Ile 275 280 285 ACC GGA GGG TAC CTC CCG CTT GCT GCG ACA CTC ACA ACG GAC GAG GTG 912 Thr Gly Gly Tyr Leu Pro Leu Ala Ala Thr Leu Thr Thr Asp Glu Val 290 295 300 TTC AAT GCC TTT TTA GGT GAG TTC GGG GAG GCA AAG CAC TTT TAC CAC 960 Phe Asn Ala Phe Leu Gly Glu Phe Gly Glu Ala Lys His Phe Tyr His 305 310 315 320 GGG CAC ACC TAC ACT GGA AAT AAC CTC GCC TGT TCC GTT GCA CTC GCA 1008 Gly His Thr Tyr Thr Gly Asn Asn Leu Ala Cys Ser Val Ala Leu Ala 325 330 335 AAC TTA GAA GTT TTT GAG GAA GAA AGA ACT TTA GAG AAG CTC CAA CCA 1056 Asn Leu Glu Val Phe Glu Glu Glu Arg Thr Leu Glu Lys Leu Gln Pro 340 345 350 AAG ATA AAG CTT TTA AAG GAA AGG CTT CAG GAG TTC TGG GAA CTC AAG 1104 Lys Ile Lys Leu Leu Lys Glu Arg Leu Gln Glu Phe Trp Glu Leu Lys 355 360 365 CAC GTT GGA GAT GTT AGA CAG CTA GGT TTT ATG GCT GGA ATA GAG CTG 1152 His Val Gly Asp Val Arg Gln Leu Gly Phe Met Ala Gly Ile Glu Leu 370 375 380 GTG AAG GAC AAA GAA AAG GGA GAA CCT TTC CCT TAC GGT GAA AGG ACG 1200 Val Lys Asp Lys Glu Lys Gly Glu Pro Phe Pro Tyr Gly Glu Arg Thr 385 390 395 400 GGA TTT AAG GTG GCT TAC AAG TGC AGG GAA AAA GGG GTG TTT TTG AGA 1248 Gly Phe Lys Val Ala Tyr Lys Cys Arg Glu Lys Gly Val Phe Leu Arg 405 410 415 CCG CTC GGA GAC GTT ATG GTA TTG ATG ATG CCT CTT GTA ATA GAG GAA 1296 Pro Leu Gly Asp Val Met Val Leu Met Met Pro Leu Val Ile Glu Glu 420 425 430 GAC GAA ATG AAC TAC GTT ATT GAT ACA CTT AAA TGG GCA ATT AAA GAG 1344 Asp Glu Met Asn Tyr Val Ile Asp Thr Leu Lys Trp Ala Ile Lys Glu 435 440 445 CTT GAA AAA GAG GTG TAG 1362 Leu Glu Lys Glu Val End 450 1032 NUCLEOTIDES NUCLEIC ACID SINGLE LINEAR GENOMIC DNA 20 ATG ACA TAC TTA ATG AAC AAT TAC GCA AGG TTG CCC GTA AAG TTT GTA 48 Met Thr Tyr Leu Met Asn Asn Tyr Ala Arg Leu Pro Val Lys Phe Val 5 10 15 AGG GGA AAA GGT GTT TAC CTG TAC GAT GAG GAA GGA AAG GAG TAT CTT 96 Arg Gly Lys Gly Val Tyr Leu Tyr Asp Glu Glu Gly Lys Glu Tyr Leu 20 25 30 GAC TTT GTC TCC GGT ATA GGC GTC AAC TCC CTC GGT CAC GCT TAC CCA 144 Asp Phe Val Ser Gly Ile Gly Val Asn Ser Leu Gly His Ala Tyr Pro 35 40 45 AAA CTC ACA GAA GCT CTA AAA GAA CAG GTT GAG AAA CTC CTC CAC GTT 192 Lys Leu Thr Glu Ala Leu Lys Glu Gln Val Glu Lys Leu Leu His Val 50 55 60 TCA AAT CTT TAC GAA AAC CCG TGG CAG GAA GAA CTG GCT CAC AAA CTT 240 Ser Asn Leu Tyr Glu Asn Pro Trp Gln Glu Glu Leu Ala His Lys Leu 65 70 75 80 GTA AAA CAC TTC TGG ACA GAA GGG AAG GTA TTT TTC GCA AAC AGC GGA 288 Val Lys His Phe Trp Thr Glu Gly Lys Val Phe Phe Ala Asn Ser Gly 85 90 95 ACG GAA AGT GTA GAG GCG GCT ATA AAG CTC GCA AGG AAG TAC TGG AGG 336 Thr Glu Ser Val Glu Ala Ala Ile Lys Leu Ala Arg Lys Tyr Trp Arg 100 105 110 GAT AAA GGA AAG AAC AAG TGG AAG TTT ATA TCC TTT GAA AAC TCT TTC 384 Asp Lys Gly Lys Asn Lys Trp Lys Phe Ile Ser Phe Glu Asn Ser Phe 115 120 125 CAC GGG AGA ACC TAC GGT AGC CTC TCC GCA ACG GGA CAG CCA AAG TTC 432 His Gly Arg Thr Tyr Gly Ser Leu Ser Ala Thr Gly Gln Pro Lys Phe 130 135 140 CAC AAA GGC TTT GAA CCT CTA GTT CCT GGA TTT TCT TAC GCA AAG CTG 480 His Lys Gly Phe Glu Pro Leu Val Pro Gly Phe Ser Tyr Ala Lys Leu 145 150 155 160 AAC GAT ATA GAC AGC GTT TAC AAA CTC CTA GAC GAG GAA ACC GCG GGG 528 Asn Asp Ile Asp Ser Val Tyr Lys Leu Leu Asp Glu Glu Thr Ala Gly 165 170 175 ATA ATT ATT GAA GTT ATA CAA GGA GAG GGC GGA GTA AAC GAG GCG AGT 576 Ile Ile Ile Glu Val Ile Gln Gly Glu Gly Gly Val Asn Glu Ala Ser 180 185 190 GAG GAT TTT CTA AGT AAA CTC CAG GAA ATT TGT AAA GAA AAA GAT GTG 624 Glu Asp Phe Leu Ser Lys Leu Gln Glu Ile Cys Lys Glu Lys Asp Val 195 200 205 CTC TTA ATT ATA GAC GAA GTG CAA ACG GGA ATA GGA AGG ACC GGG GAA 672 Leu Leu Ile Ile Asp Glu Val Gln Thr Gly Ile Gly Arg Thr Gly Glu 210 215 220 TTC TAC GCA TAT CAA CAC TTC AAT CTA AAA CCG GAC GTA ATT GCG CTT 720 Phe Tyr Ala Tyr Gln His Phe Asn Leu Lys Pro Asp Val Ile Ala Leu 225 230 235 240 GCG AAG GGA CTC GGA GGA GGT GTG CCA ATA GGT GCC ATC CTT GCA AGG 768 Ala Lys Gly Leu Gly Gly Gly Val Pro Ile Gly Ala Ile Leu Ala Arg 245 250 255 GAA GAA GTG GCC CAG AGC TTT ACT CCC GGC TCC CAC GGC TCT ACC TTC 816 Glu Glu Val Ala Gln Ser Phe Thr Pro Gly Ser His Gly Ser Thr Phe 260 265 270 GGA GGA AAC CCC TTA GCC TGC AGG GCG GGA ACA GTG GTA GTA GAT GAA 864 Gly Gly Asn Pro Leu Ala Cys Arg Ala Gly Thr Val Val Val Asp Glu 275 280 285 GTT GAA AAA CTC CTG CCT CAC GTA AGG GAA GTG GGG AAT TAC TTC AAA 912 Val Glu Lys Leu Leu Pro His Val Arg Glu Val Gly Asn Tyr Phe Lys 290 295 300 GAA AAA CTG AAG GAA CTC GGC AAA GGA AAG GTA AAG GGA AGA GGA TTG 960 Glu Lys Leu Lys Glu Leu Gly Lys Gly Lys Val Lys Gly Arg Gly Leu 305 310 315 320 ATG CTC GGT CTT GAA CTT GAA AGA GAG TGT AAA GAT TAC GTT CTC AAG 1008 Met Leu Gly Leu Glu Leu Glu Arg Glu Cys Lys Asp Tyr Val Leu Lys 325 330 335 GCT CTT GAA AGG GAC TTC TCA TAA 1032 Ala Leu Glu Arg Asp Phe Ser End 340 1197 NUCLEOTIDES NUCLEIC ACID SINGLE LINEAR GENOMIC DNA 21 ATG CGG AAA CTG GCC GAG CGG GCG CAG AAA CTG AGC CCC TCT CCC ACC 48 Met Arg Lys Leu Ala Glu Arg Ala Gln Lys Leu Ser Pro Ser Pro Thr 5 10 15 CTC TCG GTG GAC ACC AAG GCC AAG GAG CTT TTG CGG CAG GGG GAA AGG 96 Leu Ser Val Asp Thr Lys Ala Lys Glu Leu Leu Arg Gln Gly Glu Arg 20 25 30 GTC ATC AAT TTC GGG GCG GGG GAG CCG GAC TTC GAT ACA CCG GAA CAC 144 Val Ile Asn Phe Gly Ala Gly Glu Pro Asp Phe Asp Thr Pro Glu His 35 40 45 ATC AAG GAA GCG GCG AAG CGA GCT TTA GAT CAG GGC TTC ACC AAG TAC 192 Ile Lys Glu Ala Ala Lys Arg Ala Leu Asp Gln Gly Phe Thr Lys Tyr 50 55 60 ACG CCG GTG GCT GGG ATC TTA CCT CTT CGG GAG GCC ATA TGC GAG AAG 240 Thr Pro Val Ala Gly Ile Leu Pro Leu Arg Glu Ala Ile Cys Glu Lys 65 70 75 80 CTT TAC CGC GAC AAT CAA CTG GAA TAC AGC CCG AAT GAG ATC GTG GTC 288 Leu Tyr Arg Asp Asn Gln Leu Glu Tyr Ser Pro Asn Glu Ile Val Val 85 90 95 TCC TGT GGC GCC AAG CAT TCT ATT TTC AAC GCT CTG CAG GTC CTC CTG 336 Ser Cys Gly Ala Lys His Ser Ile Phe Asn Ala Leu Gln Val Leu Leu 100 105 110 GAC CCG GGG GAC GAG GTG ATA ATC CCC GTC CCC TAC TGG ACT TCC TAT 384 Asp Pro Gly Asp Glu Val Ile Ile Pro Val Pro Tyr Trp Thr Ser Tyr 115 120 125 CCG GAG CAG GTG AAG CTG GCG GGA GGG GTG CCG GTT TTC GTC CCC ACC 432 Pro Glu Gln Val Lys Leu Ala Gly Gly Val Pro Val Phe Val Pro Thr 130 135 140 TCT CCC GAG AAC GAC TTC AAG CTC AGG CCG GAA GAT CTA CGT GCG GCT 480 Ser Pro Glu Asn Asp Phe Lys Leu Arg Pro Glu Asp Leu Arg Ala Ala 145 150 155 160 GTA ACC CCG CGC ACC CGC CTT TTG ATC CTC AAT TCC CCG GCC AAC CCC 528 Val Thr Pro Arg Thr Arg Leu Leu Ile Leu Asn Ser Pro Ala Asn Pro 165 170 175 ACA GGC ACC GTT TAC CGC CGG GAG GAA CTT ATC GGC TTA GCG GAG GTA 576 Thr Gly Thr Val Tyr Arg Arg Glu Glu Leu Ile Gly Leu Ala Glu Val 180 185 190 GCC CTG GAG GCC GAC CTA TGG ATC TTG TCG GAC GAG ATC TAC GAA AAG 624 Ala Leu Glu Ala Asp Leu Trp Ile Leu Ser Asp Glu Ile Tyr Glu Lys 195 200 205 CTG ATC TAC GAC GGG ATG GAG CAC GTG AGC ATA GCC GCG CTC GAC CCG 672 Leu Ile Tyr Asp Gly Met Glu His Val Ser Ile Ala Ala Leu Asp Pro 210 215 220 GAG GTC AAA AAG CGC ACG ATT GTG GTA AAC GGT GTT TCC AAG GCT TAC 720 Glu Val Lys Lys Arg Thr Ile Val Val Asn Gly Val Ser Lys Ala Tyr 225 230 235 240 GCC ATG ACC GGT TGG CGC ATA GGT TAT GCT GCC GCT CCC CGG CCG ATA 768 Ala Met Thr Gly Trp Arg Ile Gly Tyr Ala Ala Ala Pro Arg Pro Ile 245 250 255 GCC CAG GCC ATG ACC AAC CTC CAA AGC CAC AGT ACC TCT AAC CCC ACT 816 Ala Gln Ala Met Thr Asn Leu Gln Ser His Ser Thr Ser Asn Pro Thr 260 265 270 TCC GTA GCC CAG GCG GCG GCG CTG GCC GCT CTG AAG GGG CCA CAA GAG 864 Ser Val Ala Gln Ala Ala Ala Leu Ala Ala Leu Lys Gly Pro Gln Glu 275 280 285 CCG GTG GAG AAC ATG CGC CGG GCT TTT CAA AAG CGG CGG GAT TTC ATC 912 Pro Val Glu Asn Met Arg Arg Ala Phe Gln Lys Arg Arg Asp Phe Ile 290 295 300 TGG CAG TAC CTA AAC TCC TTA CCC GGA GTG CGC TGC CCC AAA CCT TTA 960 Trp Gln Tyr Leu Asn Ser Leu Pro Gly Val Arg Cys Pro Lys Pro Leu 305 310 315 320 GGG GCC TTT TAC GTC TTT CCA GAA GTT GAG CGG GCT TTT GGG CCG CCG 1008 Gly Ala Phe Tyr Val Phe Pro Glu Val Glu Arg Ala Phe Gly Pro Pro 325 330 335 TCT AAA AGG ACG GGA AAT ACT ACC GCT AGC GAC CTG GCC CTT TTC CTC 1056 Ser Lys Arg Thr Gly Asn Thr Thr Ala Ser Asp Leu Ala Leu Phe Leu 340 345 350 CTG GAA GAG ATA AAA GTG GCC ACC GTG GCT GGG GCT GCC TTT GGG GAC 1104 Leu Glu Glu Ile Lys Val Ala Thr Val Ala Gly Ala Ala Phe Gly Asp 355 360 365 GAT CGC TAC CTG CGC TTT TCC TAC GCC CTG CGG CTG GAA GAT ATC GAA 1152 Asp Arg Tyr Leu Arg Phe Ser Tyr Ala Leu Arg Leu Glu Asp Ile Glu 370 375 380 GAG GGG ATG CAA CGG TTT AAA GAA TTG ATC GAA GCG GCA CTT TAA 1197 Glu Gly Met Gln Arg Phe Lys Glu Leu Ile Glu Ala Ala Leu End 385 390 395 1779 NUCLEOTIDES NUCLEIC ACID SINGLE LINEAR GENOMIC DNA 22 ATG TGC GGG ATA GTC GGA TAC GTA GGG AGG GAT TTA GCC CTT CCT ATA 48 Met Cys Gly Ile Val Gly Tyr Val Gly Arg Asp Leu Ala Leu Pro Ile 5 10 15 GTC CTC GGA GCT CTT GAG AGA CTC GAA TAC AGG GGT TAC GAC TCC GCG 96 Val Leu Gly Ala Leu Glu Arg Leu Glu Tyr Arg Gly Tyr Asp Ser Ala 20 25 30 GGA GTT GCC CTT ATA GAA GAC GGG AAA CTC ATA GTT GAA AAG AAG AAG 144 Gly Val Ala Leu Ile Glu Asp Gly Lys Leu Ile Val Glu Lys Lys Lys 35 40 45 GGA AAG ATA AGG GAA CTC GTT AAA GCG CTA TGG GGA AAG GAT TAC AAG 192 Gly Lys Ile Arg Glu Leu Val Lys Ala Leu Trp Gly Lys Asp Tyr Lys 50 55 60 GCT AAA ACG GGT ATA GGT CAC ACA CGC TGG GCA ACC CAC GGA AAG CCC 240 Ala Lys Thr Gly Ile Gly His Thr Arg Trp Ala Thr His Gly Lys Pro 65 70 75 80 ACG GAC GAG AAC GCC CAC CCC CAC ACC GAC GAA AAA GGT GAG TTT GCA 288 Thr Asp Glu Asn Ala His Pro His Thr Asp Glu Lys Gly Glu Phe Ala 85 90 95 GTA GTT CAC AAC GGG ATA ATA GAA AAC TAC TTA GAA CTA AAA GAG GAA 336 Val Val His Asn Gly Ile Ile Glu Asn Tyr Leu Glu Leu Lys Glu Glu 100 105 110 CTA AAG AAG GAA GGT GTA AAG TTC AGG TCC GAA ACA GAC ACA GAA GTT 384 Leu Lys Lys Glu Gly Val Lys Phe Arg Ser Glu Thr Asp Thr Glu Val 115 120 125 ATA GCC CAC CTC ATA GCG AAG AAC TAC AGG GGG GAC TTA CTG GAG GCC 432 Ile Ala His Leu Ile Ala Lys Asn Tyr Arg Gly Asp Leu Leu Glu Ala 130 135 140 GTT TTA AAA ACC GTA AAG AAA TTA AAG GGT GCT TTT GCC TTT GCG GTT 480 Val Leu Lys Thr Val Lys Lys Leu Lys Gly Ala Phe Ala Phe Ala Val 145 150 155 160 ATA ACG GTT CAC GAA CCA AAC AGA CTA ATA GGA GTG AAG CAG GGG AGT 528 Ile Thr Val His Glu Pro Asn Arg Leu Ile Gly Val Lys Gln Gly Ser 165 170 175 CCT TTA ATC GTC GGA CTC GGA GAA GGA GAA AAC TTC CTC GCT TCA GAT 576 Pro Leu Ile Val Gly Leu Gly Glu Gly Glu Asn Phe Leu Ala Ser Asp 180 185 190 ATT CCC GCA ATA CTT CCT TAC ACG AAA AAG ATT ATT GTT CTT GAT GAC 624 Ile Pro Ala Ile Leu Pro Tyr Thr Lys Lys Ile Ile Val Leu Asp Asp 195 200 205 GGG GAA ATA GCG GAC CTG ACT CCC GAC ACT GTG AAC ATT TAC AAC TTT 672 Gly Glu Ile Ala Asp Leu Thr Pro Asp Thr Val Asn Ile Tyr Asn Phe 210 215 220 GAG GGA GAG CCC GTT TCA AAG GAA GTA ATG ATT ACG CCC TGG GAT CTT 720 Glu Gly Glu Pro Val Ser Lys Glu Val Met Ile Thr Pro Trp Asp Leu 225 230 235 240 GTT TCT GCG GAA AAG GGT GGT TTT AAA CAC TTC ATG CTA AAA GAG ATA 768 Val Ser Ala Glu Lys Gly Gly Phe Lys His Phe Met Leu Lys Glu Ile 245 250 255 TAC GAA CAG CCC AAA GCC ATA AAC GAC ACA CTC AAG GGT TTC CTC TCA 816 Tyr Glu Gln Pro Lys Ala Ile Asn Asp Thr Leu Lys Gly Phe Leu Ser 260 265 270 ACC GAA GAC GCA ATA CCC TTT AAG TTA AAA GAC TTC AGA AGG GTT TTA 864 Thr Glu Asp Ala Ile Pro Phe Lys Leu Lys Asp Phe Arg Arg Val Leu 275 280 285 ATA ATA GCG TGC GGG ACC TCT TAC CAC GCG GGC TTC GTC GGA AAG TAC 912 Ile Ile Ala Cys Gly Thr Ser Tyr His Ala Gly Phe Val Gly Lys Tyr 290 295 300 TGG ATA GAG AGA TTT GCA GGT GTT CCC ACA GAG GTA ATT TAC GCT TCG 960 Trp Ile Glu Arg Phe Ala Gly Val Pro Thr Glu Val Ile Tyr Ala Ser 305 310 315 320 GAA TTC AGG TAT GCG GAC GTT CCC GTT TCG GAC AAG GAT ATC GTT ATC 1008 Glu Phe Arg Tyr Ala Asp Val Pro Val Ser Asp Lys Asp Ile Val Ile 325 330 335 GGA ATT TCC CAG TCA GGA GAG ACC GCT GAC ACA AAG TTT GCC CTT CAG 1056 Gly Ile Ser Gln Ser Gly Glu Thr Ala Asp Thr Lys Phe Ala Leu Gln 340 345 350 TCC GCA AAG GAA AAG GGA GCC TTT ACC GTG GGA CTC GTA AAC GTA GTG 1104 Ser Ala Lys Glu Lys Gly Ala Phe Thr Val Gly Leu Val Asn Val Val 355 360 365 GGA AGT GCC ATA GAC AGG GAG TCG GAC TTT TCC CTT CAC ACA CAT GCG 1152 Gly Ser Ala Ile Asp Arg Glu Ser Asp Phe Ser Leu His Thr His Ala 370 375 380 GGA CCC GAA ATA GGC GTG GCG GCT ACA AAG ACC TTC ACC GCA CAG TTC 1200 Gly Pro Glu Ile Gly Val Ala Ala Thr Lys Thr Phe Thr Ala Gln Phe 385 390 395 400 ACC GCA CTC TAC GCC CTT TCG GTA AGG GAA AGT GAG GAG AGG GAA AAT 1248 Thr Ala Leu Tyr Ala Leu Ser Val Arg Glu Ser Glu Glu Arg Glu Asn 405 410 415 CTA ATA AGA CTC CTT GAA AAG GTT CCA TCA CTC GTT GAA CAA ACA CTG 1296 Leu Ile Arg Leu Leu Glu Lys Val Pro Ser Leu Val Glu Gln Thr Leu 420 425 430 AAC ACC GCA GAA GAA GTG GAG AAG GTA GCG GAA AAG TAC ATG AAA AAG 1344 Asn Thr Ala Glu Glu Val Glu Lys Val Ala Glu Lys Tyr Met Lys Lys 435 440 445 AAA AAC ATG CTT TAC CTC GGA AGG TAC TTA AAT TAC CCC ATA GCG CTG 1392 Lys Asn Met Leu Tyr Leu Gly Arg Tyr Leu Asn Tyr Pro Ile Ala Leu 450 455 460 GAG GGA GCT CTT AAA CTT AAA GAA ATT TCT TAC ATA CAC GCG GAA GGT 1440 Glu Gly Ala Leu Lys Leu Lys Glu Ile Ser Tyr Ile His Ala Glu Gly 465 470 475 480 TAT CCC GCA GGG GAG ATG AAG CAC GGT CCC ATA GCC CTC ATA GAC GAA 1488 Tyr Pro Ala Gly Glu Met Lys His Gly Pro Ile Ala Leu Ile Asp Glu 485 490 495 AAC ATG CCG GTT GTG GTA ATC GCA CCG AAA GAC AGG GTT TAC GAG AAG 1536 Asn Met Pro Val Val Val Ile Ala Pro Lys Asp Arg Val Tyr Glu Lys 500 505 510 ATA CTC TCA AAC GTA GAA GAG GTT CTC GCA AGA AAG GGA AGG GTT ATT 1584 Ile Leu Ser Asn Val Glu Glu Val Leu Ala Arg Lys Gly Arg Val Ile 515 520 525 TCT GTA GGC TTT AAA GGA GAC GAA ACT CTC AAA AGC AAA TCC GAG AGC 1632 Ser Val Gly Phe Lys Gly Asp Glu Thr Leu Lys Ser Lys Ser Glu Ser 530 535 540 GTT ATG GAA ATC CCG AAG GCA GAA GAA CCG ATA ACT CCT TTC TTG ACG 1680 Val Met Glu Ile Pro Lys Ala Glu Glu Pro Ile Thr Pro Phe Leu Thr 545 550 555 560 GTA ATA CCC CTG CAA CTC TTT GCC TAC TTT ATA GCG AGC AAA CTG GGA 1728 Val Ile Pro Leu Gln Leu Phe Ala Tyr Phe Ile Ala Ser Lys Leu Gly 565 570 575 CTG GAT GTG GAT CAG CCG AGA AAT CTC GCC AAA ACG GTC ACG GTG GAA 1776 Leu Asp Val Asp Gln Pro Arg Asn Leu Ala Lys Thr Val Thr Val Glu 580 585 590 TAA 1779 End 1065 NUCLEOTIDES NUCLEIC ACID SINGLE LINEAR GENOMIC DNA 23 ATG ATA CCC CAG AGG ATT AAG GAA CTT GAA GCT TAC AAG ACG GAG GTC 48 Met Ile Pro Gln Arg Ile Lys Glu Leu Glu Ala Tyr Lys Thr Glu Val 5 10 15 ACT CCC GCC TCC GTC AGG CTT TCC TCT AAC GAA TTC CCC TAC GAC TTT 96 Thr Pro Ala Ser Val Arg Leu Ser Ser Asn Glu Phe Pro Tyr Asp Phe 20 25 30 CCC GAG GAG ATA AAA CAA AGG GCC TTA GAA GAA TTA AAA AAG GTT CCC 144 Pro Glu Glu Ile Lys Gln Arg Ala Leu Glu Glu Leu Lys Lys Val Pro 35 40 45 TTG AAC AAA TAC CCA GAC CCC GAA GCG AAA GAG TTA AAA GCG GTT CTT 192 Leu Asn Lys Tyr Pro Asp Pro Glu Ala Lys Glu Leu Lys Ala Val Leu 50 55 60 GCG GAT TTT TTC GGC GTT AAG GAA GAA AAT TTA GTT CTC GGT AAC GGT 240 Ala Asp Phe Phe Gly Val Lys Glu Glu Asn Leu Val Leu Gly Asn Gly 65 70 75 80 TCG GAC GAA CTC ATA TAC TAC CTC TCA ATA GCT ATA GGT GAA CTT TAC 288 Ser Asp Glu Leu Ile Tyr Tyr Leu Ser Ile Ala Ile Gly Glu Leu Tyr 85 90 95 ATA CCC GTT TAC ATA CCT GTT CCC ACC TTT CCC ATG TAC GAG ATA AGT 336 Ile Pro Val Tyr Ile Pro Val Pro Thr Phe Pro Met Tyr Glu Ile Ser 100 105 110 GCG AAA GTT CTC GGA AGA CCC CTC GTA AAG GTT CAA CTG GAC GAA AAC 384 Ala Lys Val Leu Gly Arg Pro Leu Val Lys Val Gln Leu Asp Glu Asn 115 120 125 TTT GAT ATA GAC TTA GAA AGA AGT ATT GAA TTA ATA GAG AAA GAA AAA 432 Phe Asp Ile Asp Leu Glu Arg Ser Ile Glu Leu Ile Glu Lys Glu Lys 130 135 140 CCC GTT CTC GGG TAC TTT GCT TAC CCA AAC AAC CCC ACG GGA AAC CTC 480 Pro Val Leu Gly Tyr Phe Ala Tyr Pro Asn Asn Pro Thr Gly Asn Leu 145 150 155 160 TTT TCC AGG GGA AAG ATT GAG GAG ATA AGA AAC AGG GGT GTT TTC TGT 528 Phe Ser Arg Gly Lys Ile Glu Glu Ile Arg Asn Arg Gly Val Phe Cys 165 170 175 GTA ATA GAC GAA GCC TAC TAT CAT TAC TCC GGA GAA ACC TTT CTG GAA 576 Val Ile Asp Glu Ala Tyr Tyr His Tyr Ser Gly Glu Thr Phe Leu Glu 180 185 190 GAC GCG CTC AAA AGG GAA GAT ACG GTA GTT TTG AGG ACA CTT TCA AAA 624 Asp Ala Leu Lys Arg Glu Asp Thr Val Val Leu Arg Thr Leu Ser Lys 195 200 205 ATC GGT ATG GCG AGT TTA AGG GTA GGG ATT TTA ATA GGG AAG GGG GAA 672 Ile Gly Met Ala Ser Leu Arg Val Gly Ile Leu Ile Gly Lys Gly Glu 210 215 220 ATC GTC TCA GAA ATT AAC AAG GTG AGA CTC CCC TTC AAC GTG ACC TAC 720 Ile Val Ser Glu Ile Asn Lys Val Arg Leu Pro Phe Asn Val Thr Tyr 225 230 235 240 CCC TCT CAG GTG ATG GCA AAA GTT CTC CTC ACG GAG GGA AGA GAA TTC 768 Pro Ser Gln Val Met Ala Lys Val Leu Leu Thr Glu Gly Arg Glu Phe 245 250 255 CTA ATG GAA AAG ATA CAG GAG GTT GTA ACA GAG CGA GAA AGG ATG TAC 816 Leu Met Glu Lys Ile Gln Glu Val Val Thr Glu Arg Glu Arg Met Tyr 260 265 270 GAC GAA ATG AAG AAA ATA GAA GGA GTT GAG GTT TTT CCG AGT AAG GCT 864 Asp Glu Met Lys Lys Ile Glu Gly Val Glu Val Phe Pro Ser Lys Ala 275 280 285 AAC TTC TTG CTT TTC AGA ACG CCT TAC CCC GCC CAC GAG GTT TAT CAG 912 Asn Phe Leu Leu Phe Arg Thr Pro Tyr Pro Ala His Glu Val Tyr Gln 290 295 300 GAG CTA CTG AAA AGG GAT GTC CTC GTC AGG AAC GTA TCT TAC ATG GAA 960 Glu Leu Leu Lys Arg Asp Val Leu Val Arg Asn Val Ser Tyr Met Glu 305 310 315 320 GGA CTC CAA AAG TGC CTC AGG GTA AGC GTA GGG AAA CCG GAA GAA AAC 1008 Gly Leu Gln Lys Cys Leu Arg Val Ser Val Gly Lys Pro Glu Glu Asn 325 330 335 AAC AAG TTT CTG GAA GCA CTG GAG GAG AGT ATA AAA TCC CTT TCA AGC 1056 Asn Lys Phe Leu Glu Ala Leu Glu Glu Ser Ile Lys Ser Leu Ser Ser 340 345 350 TCT CTT TAA 1065 Ser Leu End 912 NUCLEOTIDES NUCLEIC ACID SINGLE LINEAR GENOMIC DNA 24 ATG AAG CCG TAC GCT AAA TAT ATC TGG CTT GAC GGC AGA ATA CTT AAG 48 Met Lys Pro Tyr Ala Lys Tyr Ile Trp Leu Asp Gly Arg Ile Leu Lys 5 10 15 TGG GAA GAC GCG AAA ATA CAC GTG TTG ACT CAC GCG CTT CAC TAC GGA 96 Trp Glu Asp Ala Lys Ile His Val Leu Thr His Ala Leu His Tyr Gly 20 25 30 ACC TCT ATA TTC GAG GGA ATA AGA GGG TAT TGG AAC GGC GAT AAT TTG 144 Thr Ser Ile Phe Glu Gly Ile Arg Gly Tyr Trp Asn Gly Asp Asn Leu 35 40 45 CTC GTC TTT AGG TTA GAA GAA CAC ATC GAC CGC ATG TAC AGA TCG GCT 192 Leu Val Phe Arg Leu Glu Glu His Ile Asp Arg Met Tyr Arg Ser Ala 50 55 60 AAG ATA CTA GGC ATA AAT ATT CCG TAT ACA AGA GAG GAA GTC CGC CAA 240 Lys Ile Leu Gly Ile Asn Ile Pro Tyr Thr Arg Glu Glu Val Arg Gln 65 70 75 80 GCT GTA CTA GAG ACC ATA AAG GCT AAT AAC TTC CGA GAG GAT GTC TAC 288 Ala Val Leu Glu Thr Ile Lys Ala Asn Asn Phe Arg Glu Asp Val Tyr 85 90 95 ATA AGA CCT GTG GCG TTT GTC GCC TCG CAG ACG GTG ACG CTT GAC ATA 336 Ile Arg Pro Val Ala Phe Val Ala Ser Gln Thr Val Thr Leu Asp Ile 100 105 110 AGA AAT TTG GAA GTC TCC CTC GCG GTT ATT GTA TTC CCA TTT GGC AAA 384 Arg Asn Leu Glu Val Ser Leu Ala Val Ile Val Phe Pro Phe Gly Lys 115 120 125 TAC CTC TCG CCC AAC GGC ATT AAG GCA ACG ATT GTA AGC TGG CGT AGA 432 Tyr Leu Ser Pro Asn Gly Ile Lys Ala Thr Ile Val Ser Trp Arg Arg 130 135 140 GTA CAT AAT ACA ATG CTC CCT GTG ATG GCA AAA ATC GGC GGT ATA TAT 480 Val His Asn Thr Met Leu Pro Val Met Ala Lys Ile Gly Gly Ile Tyr 145 150 155 160 GTA AAC TCT GTA CTT GCG CTT GTA GAG GCT AGA AGC AGG GGA TTT GAC 528 Val Asn Ser Val Leu Ala Leu Val Glu Ala Arg Ser Arg Gly Phe Asp 165 170 175 GAG GCT TTA TTA ATG GAC GTT AAC GGT TAT GTT GTT GAG GGT TCT GGA 576 Glu Ala Leu Leu Met Asp Val Asn Gly Tyr Val Val Glu Gly Ser Gly 180 185 190 GAG AAT ATT TTC ATT GTC AGA GGT GGA AGG CTT TTC ACG CCG CCA GTA 624 Glu Asn Ile Phe Ile Val Arg Gly Gly Arg Leu Phe Thr Pro Pro Val 195 200 205 CAC GAA TCT ATC CTC GAG GGA ATT ACG AGG GAT ACG GTA ATA AAG CTC 672 His Glu Ser Ile Leu Glu Gly Ile Thr Arg Asp Thr Val Ile Lys Leu 210 215 220 AGC GGG GAT GTG GGA CTT CGG GTG GAG GAA AAG CCT ATT ACG AGG GAG 720 Ser Gly Asp Val Gly Leu Arg Val Glu Glu Lys Pro Ile Thr Arg Glu 225 230 235 240 GAG GTG TAT ACA GCC GAC GAG GTG TTT TTA GTA GGA ACC GCC GCA GAG 768 Glu Val Tyr Thr Ala Asp Glu Val Phe Leu Val Gly Thr Ala Ala Glu 245 250 255 ATA ACG CCA GTG GTG GAG GTT GAC GGC AGA ACA ATC GGC ACA GGC AAG 816 Ile Thr Pro Val Val Glu Val Asp Gly Arg Thr Ile Gly Thr Gly Lys 260 265 270 CCG GGC CCC ATT ACG ACA AAA ATA GCT GAG CTG TAC TCA AAC GTC GTG 864 Pro Gly Pro Ile Thr Thr Lys Ile Ala Glu Leu Tyr Ser Asn Val Val 275 280 285 AGA GGC AAA GTA GAG AAA TAC TTA AAT TGG ATC ACT CCT GTG TAT TAG 912 Arg Gly Lys Val Glu Lys Tyr Leu Asn Trp Ile Thr Pro Val Tyr End 290 295 300 414 AMINO ACIDS AMINO ACID LINEAR PROTEIN 25 Met Ile Glu Asp Pro Met Asp Trp Ala Phe Pro Arg Ile Lys Arg Leu 5 10 15 Pro Gln Tyr Val Phe Ser Leu Val Asn Glu Leu Lys Tyr Lys Leu Arg 20 25 30 Arg Glu Gly Glu Asp Val Val Asp Leu Gly Met Gly Asn Pro Asn Met 35 40 45 Pro Pro Ala Lys His Ile Ile Asp Lys Leu Cys Glu Val Ala Gln Lys 50 55 60 Pro Asn Val His Gly Tyr Ser Ala Ser Arg Gly Ile Pro Arg Leu Arg 65 70 75 80 Lys Ala Ile Cys Asn Phe Tyr Glu Glu Arg Tyr Gly Val Lys Leu Asp 85 90 95 Pro Glu Arg Glu Ala Ile Leu Thr Ile Gly Ala Lys Glu Gly Tyr Ser 100 105 110 His Leu Met Leu Ala Met Ile Ser Pro Gly Asp Thr Val Ile Val Pro 115 120 125 Asn Pro Thr Tyr Pro Ile His Tyr Tyr Ala Pro Ile Ile Ala Gly Gly 130 135 140 Glu Val His Ser Ile Pro Leu Asn Phe Ser Asp Asp Gln Asp His Gln 145 150 155 160 Glu Glu Phe Leu Arg Arg Leu Tyr Glu Ile Val Lys Thr Ala Met Pro 165 170 175 Lys Pro Lys Ala Val Val Ile Ser Phe Pro His Asn Pro Thr Thr Ile 180 185 190 Thr Val Glu Lys Asp Phe Phe Lys Glu Ile Val Lys Phe Ala Lys Glu 195 200 205 His Gly Leu Trp Ile Ile His Asp Phe Ala Tyr Ala Asp Ile Ala Phe 210 215 220 Asp Gly Tyr Lys Pro Pro Ser Ile Leu Glu Ile Glu Gly Ala Lys Asp 225 230 235 240 Val Ala Val Glu Leu Tyr Ser Met Ser Lys Gly Phe Ser Met Ala Gly 245 250 255 Trp Arg Val Ala Phe Val Val Gly Asn Glu Ile Leu Ile Lys Asn Leu 260 265 270 Ala His Leu Lys Ser Tyr Leu Asp Tyr Gly Ile Phe Thr Pro Ile Gln 275 280 285 Val Ala Ser Ile Ile Ala Leu Glu Ser Pro Tyr Glu Ile Val Glu Lys 290 295 300 Thr Ala Lys Val Tyr Gln Lys Arg Arg Asp Val Leu Val Glu Gly Leu 305 310 315 320 Asn Arg Leu Gly Trp Lys Val Lys Lys Pro Lys Ala Thr Met Phe Val 325 330 335 Trp Ala Lys Ile Pro Glu Trp Ile Asn Met Asn Ser Leu Asp Phe Ser 340 345 350 Leu Phe Leu Leu Lys Glu Ala Lys Val Ala Val Ser Pro Gly Val Gly 355 360 365 Phe Gly Gln Tyr Gly Glu Gly Tyr Val Arg Phe Ala Leu Val Glu Asn 370 375 380 Glu His Arg Ile Arg Gln Ala Ile Arg Gly Ile Arg Lys Ala Phe Arg 385 390 395 400 Lys Leu Gln Lys Glu Arg Lys Leu Glu Pro Glu Arg Ser Ala 405 410 414 373 AMINO ACIDS AMINO ACID LINEAR PROTEIN 26 Met Asp Arg Leu Glu Lys Val Ser Pro Phe Ile Val Met Asp Ile Leu 5 10 15 Ala Gln Ala Gln Lys Tyr Glu Asp Val Val His Met Glu Ile Gly Glu 20 25 30 Pro Asp Leu Glu Pro Ser Pro Lys Val Met Glu Ala Leu Glu Arg Ala 35 40 45 Val Lys Glu Lys Thr Phe Phe Tyr Thr Pro Ala Leu Gly Leu Trp Glu 50 55 60 Leu Arg Glu Arg Ile Ser Glu Phe Tyr Arg Lys Lys Tyr Ser Val Glu 65 70 75 80 Val Ser Pro Glu Arg Val Ile Val Thr Thr Gly Thr Ser Gly Ala Phe 85 90 95 Leu Val Ala Tyr Ala Val Thr Leu Asn Ala Gly Glu Lys Ile Ile Leu 100 105 110 Pro Asp Pro Ser Tyr Pro Cys Tyr Lys Asn Phe Ala Tyr Leu Leu Asp 115 120 125 Ala Gln Pro Val Phe Val Asn Val Asp Lys Glu Thr Asn Tyr Glu Val 130 135 140 Arg Lys Glu Met Ile Glu Asp Ile Asp Ala Lys Ala Leu His Ile Ser 145 150 155 160 Ser Pro Gln Asn Pro Thr Gly Thr Leu Tyr Ser Pro Glu Thr Leu Lys 165 170 175 Glu Leu Ala Glu Tyr Cys Glu Glu Lys Gly Met Tyr Phe Ile Ser Asp 180 185 190 Glu Ile Tyr His Gly Leu Val Tyr Glu Gly Arg Glu His Thr Ala Leu 195 200 205 Glu Phe Ser Asp Arg Ala Ile Val Ile Asn Gly Phe Ser Lys Tyr Phe 210 215 220 Cys Met Pro Gly Phe Arg Ile Gly Trp Met Ile Val Pro Glu Glu Leu 225 230 235 240 Val Arg Lys Ala Glu Ile Val Ile Gln Asn Val Phe Ile Ser Ala Pro 245 250 255 Thr Leu Ser Gln Tyr Ala Ala Leu Glu Ala Phe Asp Tyr Glu Tyr Leu 260 265 270 Glu Lys Val Arg Lys Thr Phe Glu Glu Arg Arg Asn Phe Leu Tyr Gly 275 280 285 Glu Leu Lys Lys Leu Phe Lys Ile Asp Ala Lys Pro Gln Gly Ala Phe 290 295 300 Tyr Val Trp Ala Asn Ile Ser Asp Tyr Ser Thr Asp Ser Tyr Glu Phe 305 310 315 320 Ala Leu Lys Leu Leu Arg Glu Ala Arg Val Ala Val Thr Pro Gly Val 325 330 335 Asp Phe Gly Lys Asn Lys Thr Lys Glu Tyr Ile Arg Phe Ala Tyr Thr 340 345 350 Arg Lys Ile Glu Glu Leu Lys Glu Gly Val Glu Arg Ile Lys Lys Phe 355 360 365 Leu Glu Lys Leu Ser 370 453 AMINO ACIDS AMINO ACID LINEAR PROTEIN 27 Met Trp Glu Leu Asp Pro Lys Thr Leu Glu Lys Trp Asp Lys Glu Tyr 5 10 15 Phe Trp His Pro Phe Thr Gln Met Lys Val Tyr Arg Glu Glu Glu Asn 20 25 30 Leu Ile Phe Glu Arg Gly Glu Gly Val Tyr Leu Trp Asp Ile Tyr Gly 35 40 45 Arg Lys Tyr Ile Asp Ala Ile Ser Ser Leu Trp Cys Asn Val His Gly 50 55 60 His Asn His Pro Lys Leu Asn Asn Ala Val Met Lys Gln Leu Cys Lys 65 70 75 80 Val Ala His Thr Thr Thr Leu Gly Ser Ser Asn Val Pro Ala Ile Leu 85 90 95 Leu Ala Lys Lys Leu Val Glu Ile Ser Pro Glu Gly Leu Asn Lys Val 100 105 110 Phe Tyr Ser Glu Asp Gly Ala Glu Ala Val Glu Ile Ala Ile Lys Met 115 120 125 Ala Tyr His Tyr Trp Lys Asn Lys Gly Val Lys Gly Lys Asn Val Phe 130 135 140 Ile Thr Leu Ser Glu Ala Tyr His Gly Asp Thr Val Gly Ala Val Ser 145 150 155 160 Val Gly Gly Ile Glu Leu Phe His Gly Thr Tyr Lys Asp Leu Leu Phe 165 170 175 Lys Thr Ile Lys Leu Pro Ser Pro Tyr Leu Tyr Cys Lys Glu Lys Tyr 180 185 190 Gly Glu Leu Cys Pro Glu Cys Thr Ala Asp Leu Leu Lys Gln Leu Glu 195 200 205 Asp Ile Leu Lys Ser Arg Glu Asp Ile Val Ala Val Ile Met Glu Ala 210 215 220 Gly Ile Gln Ala Ala Ala Gly Met Leu Pro Phe Pro Pro Gly Phe Leu 225 230 235 240 Lys Gly Val Arg Glu Leu Thr Lys Lys Tyr Asp Thr Leu Met Ile Val 245 250 255 Asp Glu Val Ala Thr Gly Phe Gly Arg Thr Gly Thr Met Phe Tyr Cys 260 265 270 Glu Gln Glu Gly Val Ser Pro Asp Phe Met Cys Leu Gly Lys Gly Ile 275 280 285 Thr Gly Gly Tyr Leu Pro Leu Ala Ala Thr Leu Thr Thr Asp Glu Val 290 295 300 Phe Asn Ala Phe Leu Gly Glu Phe Gly Glu Ala Lys His Phe Tyr His 305 310 315 320 Gly His Thr Tyr Thr Gly Asn Asn Leu Ala Cys Ser Val Ala Leu Ala 325 330 335 Asn Leu Glu Val Phe Glu Glu Glu Arg Thr Leu Glu Lys Leu Gln Pro 340 345 350 Lys Ile Lys Leu Leu Lys Glu Arg Leu Gln Glu Phe Trp Glu Leu Lys 355 360 365 His Val Gly Asp Val Arg Gln Leu Gly Phe Met Ala Gly Ile Glu Leu 370 375 380 Val Lys Asp Lys Glu Lys Gly Glu Pro Phe Pro Tyr Gly Glu Arg Thr 385 390 395 400 Gly Phe Lys Val Ala Tyr Lys Cys Arg Glu Lys Gly Val Phe Leu Arg 405 410 415 Pro Leu Gly Asp Val Met Val Leu Met Met Pro Leu Val Ile Glu Glu 420 425 430 Asp Glu Met Asn Tyr Val Ile Asp Thr Leu Lys Trp Ala Ile Lys Glu 435 440 445 Leu Glu Lys Glu Val 450 343 AMINO ACIDS AMINO ACID LINEAR PROTEIN 28 Met Thr Tyr Leu Met Asn Asn Tyr Ala Arg Leu Pro Val Lys Phe Val 5 10 15 Arg Gly Lys Gly Val Tyr Leu Tyr Asp Glu Glu Gly Lys Glu Tyr Leu 20 25 30 Asp Phe Val Ser Gly Ile Gly Val Asn Ser Leu Gly His Ala Tyr Pro 35 40 45 Lys Leu Thr Glu Ala Leu Lys Glu Gln Val Glu Lys Leu Leu His Val 50 55 60 Ser Asn Leu Tyr Glu Asn Pro Trp Gln Glu Glu Leu Ala His Lys Leu 65 70 75 80 Val Lys His Phe Trp Thr Glu Gly Lys Val Phe Phe Ala Asn Ser Gly 85 90 95 Thr Glu Ser Val Glu Ala Ala Ile Lys Leu Ala Arg Lys Tyr Trp Arg 100 105 110 Asp Lys Gly Lys Asn Lys Trp Lys Phe Ile Ser Phe Glu Asn Ser Phe 115 120 125 His Gly Arg Thr Tyr Gly Ser Leu Ser Ala Thr Gly Gln Pro Lys Phe 130 135 140 His Lys Gly Phe Glu Pro Leu Val Pro Gly Phe Ser Tyr Ala Lys Leu 145 150 155 160 Asn Asp Ile Asp Ser Val Tyr Lys Leu Leu Asp Glu Glu Thr Ala Gly 165 170 175 Ile Ile Ile Glu Val Ile Gln Gly Glu Gly Gly Val Asn Glu Ala Ser 180 185 190 Glu Asp Phe Leu Ser Lys Leu Gln Glu Ile Cys Lys Glu Lys Asp Val 195 200 205 Leu Leu Ile Ile Asp Glu Val Gln Thr Gly Ile Gly Arg Thr Gly Glu 210 215 220 Phe Tyr Ala Tyr Gln His Phe Asn Leu Lys Pro Asp Val Ile Ala Leu 225 230 235 240 Ala Lys Gly Leu Gly Gly Gly Val Pro Ile Gly Ala Ile Leu Ala Arg 245 250 255 Glu Glu Val Ala Gln Ser Phe Thr Pro Gly Ser His Gly Ser Thr Phe 260 265 270 Gly Gly Asn Pro Leu Ala Cys Arg Ala Gly Thr Val Val Val Asp Glu 275 280 285 Val Glu Lys Leu Leu Pro His Val Arg Glu Val Gly Asn Tyr Phe Lys 290 295 300 Glu Lys Leu Lys Glu Leu Gly Lys Gly Lys Val Lys Gly Arg Gly Leu 305 310 315 320 Met Leu Gly Leu Glu Leu Glu Arg Glu Cys Lys Asp Tyr Val Leu Lys 325 330 335 Ala Leu Glu Arg Asp Phe Ser 340 398 AMINO ACIDS AMINO ACID LINEAR PROTEIN 29 Met Arg Lys Leu Ala Glu Arg Ala Gln Lys Leu Ser Pro Ser Pro Thr 5 10 15 Leu Ser Val Asp Thr Lys Ala Lys Glu Leu Leu Arg Gln Gly Glu Arg 20 25 30 Val Ile Asn Phe Gly Ala Gly Glu Pro Asp Phe Asp Thr Pro Glu His 35 40 45 Ile Lys Glu Ala Ala Lys Arg Ala Leu Asp Gln Gly Phe Thr Lys Tyr 50 55 60 Thr Pro Val Ala Gly Ile Leu Pro Leu Arg Glu Ala Ile Cys Glu Lys 65 70 75 80 Leu Tyr Arg Asp Asn Gln Leu Glu Tyr Ser Pro Asn Glu Ile Val Val 85 90 95 Ser Cys Gly Ala Lys His Ser Ile Phe Asn Ala Leu Gln Val Leu Leu 100 105 110 Asp Pro Gly Asp Glu Val Ile Ile Pro Val Pro Tyr Trp Thr Ser Tyr 115 120 125 Pro Glu Gln Val Lys Leu Ala Gly Gly Val Pro Val Phe Val Pro Thr 130 135 140 Ser Pro Glu Asn Asp Phe Lys Leu Arg Pro Glu Asp Leu Arg Ala Ala 145 150 155 160 Val Thr Pro Arg Thr Arg Leu Leu Ile Leu Asn Ser Pro Ala Asn Pro 165 170 175 Thr Gly Thr Val Tyr Arg Arg Glu Glu Leu Ile Gly Leu Ala Glu Val 180 185 190 Ala Leu Glu Ala Asp Leu Trp Ile Leu Ser Asp Glu Ile Tyr Glu Lys 195 200 205 Leu Ile Tyr Asp Gly Met Glu His Val Ser Ile Ala Ala Leu Asp Pro 210 215 220 Glu Val Lys Lys Arg Thr Ile Val Val Asn Gly Val Ser Lys Ala Tyr 225 230 235 240 Ala Met Thr Gly Trp Arg Ile Gly Tyr Ala Ala Ala Pro Arg Pro Ile 245 250 255 Ala Gln Ala Met Thr Asn Leu Gln Ser His Ser Thr Ser Asn Pro Thr 260 265 270 Ser Val Ala Gln Ala Ala Ala Leu Ala Ala Leu Lys Gly Pro Gln Glu 275 280 285 Pro Val Glu Asn Met Arg Arg Ala Phe Gln Lys Arg Arg Asp Phe Ile 290 295 300 Trp Gln Tyr Leu Asn Ser Leu Pro Gly Val Arg Cys Pro Lys Pro Leu 305 310 315 320 Gly Ala Phe Tyr Val Phe Pro Glu Val Glu Arg Ala Phe Gly Pro Pro 325 330 335 Ser Lys Arg Thr Gly Asn Thr Thr Ala Ser Asp Leu Ala Leu Phe Leu 340 345 350 Leu Glu Glu Ile Lys Val Ala Thr Val Ala Gly Ala Ala Phe Gly Asp 355 360 365 Asp Arg Tyr Leu Arg Phe Ser Tyr Ala Leu Arg Leu Glu Asp Ile Glu 370 375 380 Glu Gly Met Gln Arg Phe Lys Glu Leu Ile Glu Ala Ala Leu 385 390 395 592 AMINO ACIDS AMINO ACID LINEAR PROTEIN 30 Met Cys Gly Ile Val Gly Tyr Val Gly Arg Asp Leu Ala Leu Pro Ile 5 10 15 Val Leu Gly Ala Leu Glu Arg Leu Glu Tyr Arg Gly Tyr Asp Ser Ala 20 25 30 Gly Val Ala Leu Ile Glu Asp Gly Lys Leu Ile Val Glu Lys Lys Lys 35 40 45 Gly Lys Ile Arg Glu Leu Val Lys Ala Leu Trp Gly Lys Asp Tyr Lys 50 55 60 Ala Lys Thr Gly Ile Gly His Thr Arg Trp Ala Thr His Gly Lys Pro 65 70 75 80 Thr Asp Glu Asn Ala His Pro His Thr Asp Glu Lys Gly Glu Phe Ala 85 90 95 Val Val His Asn Gly Ile Ile Glu Asn Tyr Leu Glu Leu Lys Glu Glu 100 105 110 Leu Lys Lys Glu Gly Val Lys Phe Arg Ser Glu Thr Asp Thr Glu Val 115 120 125 Ile Ala His Leu Ile Ala Lys Asn Tyr Arg Gly Asp Leu Leu Glu Ala 130 135 140 Val Leu Lys Thr Val Lys Lys Leu Lys Gly Ala Phe Ala Phe Ala Val 145 150 155 160 Ile Thr Val His Glu Pro Asn Arg Leu Ile Gly Val Lys Gln Gly Ser 165 170 175 Pro Leu Ile Val Gly Leu Gly Glu Gly Glu Asn Phe Leu Ala Ser Asp 180 185 190 Ile Pro Ala Ile Leu Pro Tyr Thr Lys Lys Ile Ile Val Leu Asp Asp 195 200 205 Gly Glu Ile Ala Asp Leu Thr Pro Asp Thr Val Asn Ile Tyr Asn Phe 210 215 220 Glu Gly Glu Pro Val Ser Lys Glu Val Met Ile Thr Pro Trp Asp Leu 225 230 235 240 Val Ser Ala Glu Lys Gly Gly Phe Lys His Phe Met Leu Lys Glu Ile 245 250 255 Tyr Glu Gln Pro Lys Ala Ile Asn Asp Thr Leu Lys Gly Phe Leu Ser 260 265 270 Thr Glu Asp Ala Ile Pro Phe Lys Leu Lys Asp Phe Arg Arg Val Leu 275 280 285 Ile Ile Ala Cys Gly Thr Ser Tyr His Ala Gly Phe Val Gly Lys Tyr 290 295 300 Trp Ile Glu Arg Phe Ala Gly Val Pro Thr Glu Val Ile Tyr Ala Ser 305 310 315 320 Glu Phe Arg Tyr Ala Asp Val Pro Val Ser Asp Lys Asp Ile Val Ile 325 330 335 Gly Ile Ser Gln Ser Gly Glu Thr Ala Asp Thr Lys Phe Ala Leu Gln 340 345 350 Ser Ala Lys Glu Lys Gly Ala Phe Thr Val Gly Leu Val Asn Val Val 355 360 365 Gly Ser Ala Ile Asp Arg Glu Ser Asp Phe Ser Leu His Thr His Ala 370 375 380 Gly Pro Glu Ile Gly Val Ala Ala Thr Lys Thr Phe Thr Ala Gln Phe 385 390 395 400 Thr Ala Leu Tyr Ala Leu Ser Val Arg Glu Ser Glu Glu Arg Glu Asn 405 410 415 Leu Ile Arg Leu Leu Glu Lys Val Pro Ser Leu Val Glu Gln Thr Leu 420 425 430 Asn Thr Ala Glu Glu Val Glu Lys Val Ala Glu Lys Tyr Met Lys Lys 435 440 445 Lys Asn Met Leu Tyr Leu Gly Arg Tyr Leu Asn Tyr Pro Ile Ala Leu 450 455 460 Glu Gly Ala Leu Lys Leu Lys Glu Ile Ser Tyr Ile His Ala Glu Gly 465 470 475 480 Tyr Pro Ala Gly Glu Met Lys His Gly Pro Ile Ala Leu Ile Asp Glu 485 490 495 Asn Met Pro Val Val Val Ile Ala Pro Lys Asp Arg Val Tyr Glu Lys 500 505 510 Ile Leu Ser Asn Val Glu Glu Val Leu Ala Arg Lys Gly Arg Val Ile 515 520 525 Ser Val Gly Phe Lys Gly Asp Glu Thr Leu Lys Ser Lys Ser Glu Ser 530 535 540 Val Met Glu Ile Pro Lys Ala Glu Glu Pro Ile Thr Pro Phe Leu Thr 545 550 555 560 Val Ile Pro Leu Gln Leu Phe Ala Tyr Phe Ile Ala Ser Lys Leu Gly 565 570 575 Leu Asp Val Asp Gln Pro Arg Asn Leu Ala Lys Thr Val Thr Val Glu 580 585 590 354 AMINO ACIDS AMINO ACID LINEAR PROTEIN 31 Met Ile Pro Gln Arg Ile Lys Glu Leu Glu Ala Tyr Lys Thr Glu Val 5 10 15 Thr Pro Ala Ser Val Arg Leu Ser Ser Asn Glu Phe Pro Tyr Asp Phe 20 25 30 Pro Glu Glu Ile Lys Gln Arg Ala Leu Glu Glu Leu Lys Lys Val Pro 35 40 45 Leu Asn Lys Tyr Pro Asp Pro Glu Ala Lys Glu Leu Lys Ala Val Leu 50 55 60 Ala Asp Phe Phe Gly Val Lys Glu Glu Asn Leu Val Leu Gly Asn Gly 65 70 75 80 Ser Asp Glu Leu Ile Tyr Tyr Leu Ser Ile Ala Ile Gly Glu Leu Tyr 85 90 95 Ile Pro Val Tyr Ile Pro Val Pro Thr Phe Pro Met Tyr Glu Ile Ser 100 105 110 Ala Lys Val Leu Gly Arg Pro Leu Val Lys Val Gln Leu Asp Glu Asn 115 120 125 Phe Asp Ile Asp Leu Glu Arg Ser Ile Glu Leu Ile Glu Lys Glu Lys 130 135 140 Pro Val Leu Gly Tyr Phe Ala Tyr Pro Asn Asn Pro Thr Gly Asn Leu 145 150 155 160 Phe Ser Arg Gly Lys Ile Glu Glu Ile Arg Asn Arg Gly Val Phe Cys 165 170 175 Val Ile Asp Glu Ala Tyr Tyr His Tyr Ser Gly Glu Thr Phe Leu Glu 180 185 190 Asp Ala Leu Lys Arg Glu Asp Thr Val Val Leu Arg Thr Leu Ser Lys 195 200 205 Ile Gly Met Ala Ser Leu Arg Val Gly Ile Leu Ile Gly Lys Gly Glu 210 215 220 Ile Val Ser Glu Ile Asn Lys Val Arg Leu Pro Phe Asn Val Thr Tyr 225 230 235 240 Pro Ser Gln Val Met Ala Lys Val Leu Leu Thr Glu Gly Arg Glu Phe 245 250 255 Leu Met Glu Lys Ile Gln Glu Val Val Thr Glu Arg Glu Arg Met Tyr 260 265 270 Asp Glu Met Lys Lys Ile Glu Gly Val Glu Val Phe Pro Ser Lys Ala 275 280 285 Asn Phe Leu Leu Phe Arg Thr Pro Tyr Pro Ala His Glu Val Tyr Gln 290 295 300 Glu Leu Leu Lys Arg Asp Val Leu Val Arg Asn Val Ser Tyr Met Glu 305 310 315 320 Gly Leu Gln Lys Cys Leu Arg Val Ser Val Gly Lys Pro Glu Glu Asn 325 330 335 Asn Lys Phe Leu Glu Ala Leu Glu Glu Ser Ile Lys Ser Leu Ser Ser 340 345 350 Ser Leu 303 AMINO ACIDS AMINO ACID LINEAR PROTEIN 32 Met Lys Pro Tyr Ala Lys Tyr Ile Trp Leu Asp Gly Arg Ile Leu Lys 5 10 15 Trp Glu Asp Ala Lys Ile His Val Leu Thr His Ala Leu His Tyr Gly 20 25 30 Thr Ser Ile Phe Glu Gly Ile Arg Gly Tyr Trp Asn Gly Asp Asn Leu 35 40 45 Leu Val Phe Arg Leu Glu Glu His Ile Asp Arg Met Tyr Arg Ser Ala 50 55 60 Lys Ile Leu Gly Ile Asn Ile Pro Tyr Thr Arg Glu Glu Val Arg Gln 65 70 75 80 Ala Val Leu Glu Thr Ile Lys Ala Asn Asn Phe Arg Glu Asp Val Tyr 85 90 95 Ile Arg Pro Val Ala Phe Val Ala Ser Gln Thr Val Thr Leu Asp Ile 100 105 110 Arg Asn Leu Glu Val Ser Leu Ala Val Ile Val Phe Pro Phe Gly Lys 115 120 125 Tyr Leu Ser Pro Asn Gly Ile Lys Ala Thr Ile Val Ser Trp Arg Arg 130 135 140 Val His Asn Thr Met Leu Pro Val Met Ala Lys Ile Gly Gly Ile Tyr 145 150 155 160 Val Asn Ser Val Leu Ala Leu Val Glu Ala Arg Ser Arg Gly Phe Asp 165 170 175 Glu Ala Leu Leu Met Asp Val Asn Gly Tyr Val Val Glu Gly Ser Gly 180 185 190 Glu Asn Ile Phe Ile Val Arg Gly Gly Arg Leu Phe Thr Pro Pro Val 195 200 205 His Glu Ser Ile Leu Glu Gly Ile Thr Arg Asp Thr Val Ile Lys Leu 210 215 220 Ser Gly Asp Val Gly Leu Arg Val Glu Glu Lys Pro Ile Thr Arg Glu 225 230 235 240 Glu Val Tyr Thr Ala Asp Glu Val Phe Leu Val Gly Thr Ala Ala Glu 245 250 255 Ile Thr Pro Val Val Glu Val Asp Gly Arg Thr Ile Gly Thr Gly Lys 260 265 270 Pro Gly Pro Ile Thr Thr Lys Ile Ala Glu Leu Tyr Ser Asn Val Val 275 280 285 Arg Gly Lys Val Glu Lys Tyr Leu Asn Trp Ile Thr Pro Val Tyr 290 295 300 

We claim:
 1. An isolated polynucleotide having at least 70% identity to a polynucleotide encoding an enzyme having aminotransferase and/or transaminase activity, wherein said polynucleotide is amplified using an oligonucleotide primer pair selected from: 5′ CCGAGAATTCATTAAAGAGGAGAAATTAACTATGATTGAAGACCCTATGGAC (SEQ. ID NO:1) and 3′ CGAAGATCTTTAGCACTTCTCTCAGGTTC; (SEQ. ID NO:2) 5′ CCGAGAATTCATTAAAGAGGAGAAATTAACTATGGACAGGCTTGAAAAAGTA (SEQ ID NO:3) and 3′ CGGAAGATCTTCAGCTAAGCTTCTCTAAGAA; (SEQ ID NO:4) 5′ CCGACAATTGATTAAAGAGGAGAAATTAACTATGTGGGAATTAGACCCTAAA (SEQ ID NO:5) and 3′ CGGAGGATCCCTACACCTCTTTTTCAAGCT; (SEQ ID NO:6) 5′ CCGACAATTGATTAAAGAGGAGAAATTAACTATGACATACTTAATGAACAAT (SEQ ID NO:7) and 3′ CGGAAGATCTTTATGAGAAGTCCCTTTCAAG; (SEQ ID NO:8) 5′ CCGAGAATTCATTAAAGAGGAGAAATTAACTATGCGGAAACTGGCCGAGCGG (SEQ ID NO:9) and 3′ CGGAGGATCCTTAAAGTGCCGCTTCGATCAA; (SEQ ID NO:10) 5′ CCGACAATTGATTAAAGAGGAGAAATTAACTATGTGCGGGATAGTCGGATAC (SEQ ID NO:11) and 3′ CGGAAGATCTTTATTCCACCGTGACCGTTTT; (SEQ ID NO:12) 5′ CCGACAATTGATTAAAGAGGAGAAATTAACTATGATACCCCAGAGGATTAAG (SEQ ID NO:13) and 3′ CGGAAGATCTTTAAAGAGAGCTTGAAAGGGA; (SEQ ID NO:14) 5′ CCGAGAATTCATTAAAGAGGAGAAATTAACIATGAAGCCGTACGCTAAATAT (SEQ ID NO:15) and 3′ CGGAAGATCTCTAATACACAGGAGTGATCCA; (SEQ ID NO:16) 5′ CCGAGAATTCATTAGAGGAGAAATTAACTATGGCAGTCAAAGTGCGGCCT (SEQ ID NO:33) and 3′ -CGGAGGATCCTTATCCAAAGCTTCCAGGAAG; (SEQ ID NO:34) or 5′ CCGAGAATTCATTAAAGAGGAGAAATTAACTATGAGAAAAGGACTTGCAAGT (SEQ ID NO:37) and 3′ CGGAGGATCCTTAGATCTCTTCAAGGGCTTT. (SEQ ID NO:38) 